Compositions, methods, and systems for the synthesis and use of imaging agents

ABSTRACT

The present invention generally relates to novel synthetic methods, systems, kits, salts, and precursors useful in medical imaging. In some embodiments, the present invention provides compositions comprising an imaging agent precursor, which may be formed using the synthetic methods described herein. An imaging agent may be converted to an imaging agent using the methods described herein. In some cases, the imaging agent is enriched in  18 F. In some cases, an imaging agent including salt forms (e.g., ascorbate salt) may be used to image an area of interest in a subject, including, but not limited to, the heart, cardiovascular system, cardiac vessels, brain, and other organs.

RELATED APPLICATIONS

The present application is a continuation and claims priority to U.S.Ser. No. 15/170,848, filed Jun. 1, 2016, entitled “Compositions,Methods, and Systems for the Synthesis and Use of Imaging Agents”, whichis a divisional and claims priority to U.S. Ser. No. 13/697,287, filedFeb. 27, 2013, entitled “Compositions, Methods, and Systems for theSynthesis and Use of Imaging Agents”, which is a national stage filingunder 35 U.S.C. §371 of International Application No. PCT/US2011/036142,filed on May 5, 2011, entitled “Compositions, Methods, and Systems forthe Synthesis and Use of Imaging Agents”, each of which is incorporatedherein by reference. International Application No. PCT/US2011/036142claims priority under 35 U.S.C. §119(e) to U.S. provisional application,U.S. Ser. No. 61/333,618, filed May 11, 2010, entitled “Compositions,Methods, and Systems For Imaging Heart Failure”; U.S. provisionalapplication, U.S. Ser. No. 61/405,524, filed Oct. 21, 2010, entitled“Compositions, Methods, and Systems For Imaging Heart Failure”; and U.S.provisional application, U.S. Ser. No. 61/405,571, filed Oct. 21, 2010,entitled “Synthetic Methods, Salts, and Compositions for Imaging”, eachof which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to systems, compositions, methods, andapparatuses for synthesizing imaging agents and precursors thereof.

BACKGROUND OF THE INVENTION

Heart failure (HF) is defined as the inability of the heart to supplyperipheral organs with sufficient blood flow. It may be characterized bya hyperadrenergic state whereby increased systemic levels ofnorepinephrine (NE) and increased local spillover of catecholaminesoccurs. The condition afflicts increasingly more people each year and isa common end-stage of many cardiac diseases and conditions includingmyocardial infarction, pressure/volume overload, viral myocarditis,toxic cardiomyopathy, valve failure, and other abnormalities. Theresultant myocardial damage, in conjunction with neurohormonal andcytokine activation, stimulates chamber remodeling which is the initialphase of HF development. The remodeling process results in decreasedoverall myocardial efficiency and eventual progression to clinical HF.To date, no cure for the condition exists, thus early diagnosis is a keyfactor in its management and long-term prognosis. An imaging agent thatidentifies subjects in early HF would thus enable treatment applicationand life-style improvements for patients living with the condition.

Accordingly, improved methods, systems, and apparatuses are needed forthe synthesis and administration of imaging agents (e.g., for imagingthe heart). In addition, while numerous synthetic methods exist for thepreparation of PET-based imaging agents, they generally require multiplesynthetic (e.g., labeling a compound with an imaging moiety) and/orpurification steps, have low chemical fidelity, and/or have low chemicalefficiency. Improved synthetic methods and compositions are thus neededfor preparing such compounds.

SUMMARY OF THE INVENTION

The invention provides, in a broad sense, methods for synthesizingimaging agents and their precursors, compounds (including salt forms)that are imaging agent precursors or imaging agents, and methods of usethereof.

In one aspect, the invention provides compositions. In some embodiments,a composition comprises a compound comprising formula (II):

or a salt, free base, or combination thereof, wherein R¹ is alkyl,haloalkyl, alkynyl, alkenyl, heteroalkyl, cycloalkyl, aryl, heteroaryl,arylalkyl, heterocyclyl, or heteroarylalkyl, each optionallysubstituted; each R² can be the same or different and is hydrogen or anitrogen-protecting group; R³, R⁴, R⁵, and R⁶ can be the same ordifferent and are individually hydrogen, C₁-C₆ alkyl, heteroalkyl,halide, —OR⁷, —SR⁷, —N(R⁷)₂, or —C(═O)R⁸, each optionally substituted;each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, or heteroaryl,each optionally substituted; each R⁸ can be the same or different and ishydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl,heteroaryl, —OH, alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, eachoptionally substituted; m is an integer between 1 and 12, inclusive; andn is an integer between 1 and 4, inclusive.

In some embodiments, a compound of formula (II) comprises the structureof formula (IV):

or a salt, free base, or combination thereof.

In some embodiments, a compound of formula (IV) comprises formula (III):

wherein X^(⊖) is a counter anion. In some embodiments, X^(⊖) is halide,phosphate, sulfate, trifluoroacetate, toluenesulfonate, acetate,formate, citric, ascorbate, mesylate (methanesulfonate), or benzoate.

In some embodiments, a compound of formula (II) comprises the formula:

In some embodiments, for any of the composition described above, atleast one R² is not hydrogen.

In some embodiments, a compound of formula (II) comprises the formula:

or a salt, free base, or combination thereof.

In some embodiments, a compound of formula (II) comprises the formula:

or a salt, free base, or combination thereof.

In some embodiments, m is 3. In some embodiments, n is 1. In someembodiments, R³ is Br. In some embodiments, R¹ is C₁-C₆ alkyl,haloalkyl, or aryl. In some embodiments, R¹ is methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, t-butyl, pentyl, or hexyl. In someembodiments, R¹ is haloalkyl. In some embodiments, R¹ is CF₃. In someembodiments, R¹ is phenyl (Ph), optionally substituted. In someembodiments, R¹ is 4-CH₃Ph, 2,4,6-(CH₃)₃C₆H₂, or C₆H₄X, wherein X ishalide. In some embodiments, m is an integer between 1 and 10,inclusive; or between 1 and 8, inclusive; or between 1 and 6, inclusive.In some embodiments, R⁴, R⁵, and R⁶ are hydrogen; and R³ is halide(e.g., Br). In some embodiments, the composition comprises a salt of thecompound of formula (II). In some embodiments, the salt is apharmaceutically acceptable salt. In some embodiments, at least one R²is t-butyloxycarbonyl.

In one aspect, the invention provides a compound comprising formula:

or a salt thereof, wherein m is an integer between 2 and 12, inclusive.In certain embodiments, m is an integer between 3 and 12, inclusive. Inone embodiment, m is 3.

In one embodiment, the invention provides a compound having a structureof:

In one aspect, the invention provides a compound comprising formula:

or a salt, free base, or combination thereof, wherein m is an integerbetween 2 and 12, inclusive. In certain embodiments, m is an integerbetween 3 and 12, inclusive. In one embodiment, m is 3.

In one embodiment, the invention provides a compound having a structureof

or a free base, salt, or combination thereof.

In one aspect, the invention provides a compound comprising formula:

or a salt, free base, or combination thereof; wherein each R² can be thesame or different and is hydrogen or a nitrogen-protecting group; and mis an integer between 2 and 12, inclusive. In certain embodiments, m isan integer between 3 and 12, inclusive. In one embodiment, m is 3.

In certain embodiments, the invention provides a compound comprisingformula:

In certain embodiments, the invention provides a compound having astructure of

wherein R² can be the same or different and is hydrogen or anitrogen-protecting group.

In one embodiment, the invention provides a compound having a structureof

In one embodiment, the invention provides a compound having a structureof

In one aspect, the invention provides a method comprising reducing acompound comprising formula:

or a salt thereof, wherein m is an integer between 3 and 12, inclusive,with a reductant under suitable conditions to form a compoundcomprising:

or a salt, free base, or combination thereof. In one embodiment, m is 3.In one embodiment, the reductant is BH₃.

In one aspect, the invention provides a method comprising reacting acompound comprising formula:

or a salt, free base, or combination thereof, wherein m is an integerbetween 2 and 12, inclusive; under conditions suitable to form acompound comprising formula:

or a salt, free base, or combination thereof, wherein each R² can be thesame or different and is hydrogen or a nitrogen-protecting group; and mis an integer between 2 and 12, inclusive. In certain embodiments, m isan integer between 3 and 12, inclusive. In one embodiment, m is 3. Inone embodiment, the step of reacting comprises reacting a comprisingformula:

with a compound of formula:

In one embodiment, the compound comprising formula:

is of formula:

In one embodiment, the compound comprising formula:

is of formula:

In other aspects, the invention provides compositions comprising one ormore of any of the foregoing compounds, including free bases thereof,salts thereof, and combinations thereof.

In another aspect, the present invention provides methods for formingcompounds. In a first embodiment, a method comprises reacting a compoundcomprising formula (II):

or a salt, free base, or combination thereof, under conditions suitableto form a compound comprising formula (IV):

or a salt, free base, or combination thereof, wherein R¹ is alkyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,alkenyl, alkynyl, heterocyclyl, or haloalkyl, each optionallysubstituted; each R² can be the same or different and is hydrogen or anitrogen-protecting group, provided at least one R² is not hydrogen; R³,R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted; each R⁷ can be the same ordifferent and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl,aryl, heteroaryl, or heterocyclyl, each optionally substituted; each R⁸can be the same or different and is hydrogen, alkyl, heteroalkyl,cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, —OH, alkoxy,—NH₂, alkylamino, —SH, or alkylthiol, each optionally substituted; m isan integer between 1 and 12, inclusive; and n is an integer between 1and 4, inclusive.

In another embodiment, a method comprises reacting a compound comprisingformula (II):

or a salt, free base, or combination thereof, under conditions suitableto form a compound comprising formula (I):

or a salt, free base, or combination thereof, wherein R¹ is alkyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heterocyclyl,heteroarylalkyl, alkenyl, alkynyl, or haloalkyl, each optionallysubstituted; each R² can be the same or different and is hydrogen or anitrogen-protecting group; R³, R⁴, R⁵, and R⁶ can be the same ordifferent and are individually hydrogen, C₁-C₆ alkyl, heteroalkyl,halide, —OR⁷, —SR⁷, —N(R⁷)₂, or —C(═O)R⁸, each optionally substituted;each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl, or heterocyclyl,each optionally substituted; each R⁸ can be the same or different and ishydrogen, alkyl heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl,heteroaryl, —OH, alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, eachoptionally substituted; m is an integer between 1 and 12, inclusive; andn is an integer between 1 and 4, inclusive.

In some embodiments, the method further comprises reacting the compoundcomprising formula (I):

or a salt, free base, or combination thereof, provided at least one R²is not H, under conditions suitable to form a compound comprisingformula (V):

or a salt, free base, or combination thereof.

In yet another embodiment, a method comprises reacting a compoundcomprising formula (IV):

or a salt, free base, or combination thereof, under conditions suitableto form a compound comprising formula (V):

or a salt, free base, or combination thereof, wherein R¹ is alkyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl,heteroarylalkyl, alkenyl, alkynyl, or haloalkyl, each optionallysubstituted; R³, R⁴, R⁵, and R⁶ can be the same or different and areindividually hydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷,—N(R⁷)₂, or —C(═O)R⁸, each optionally substituted; each R⁷ can be thesame or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl,haloalkyl, aryl, heteroaryl, or heterocyclyl, each optionallysubstituted; each R⁸ can be the same or different and is hydrogen,alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl,heteroaryl, —OH, alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, eachoptionally substituted; m is an integer between 1 and 12, inclusive; andn is an integer between 1 and 4, inclusive.

In some embodiments, a compound of formula (II) comprises formula (IV):

or a salt, free base, or combination thereof.

In some embodiments, a compound of formula (IV) comprises formula (III):

wherein X^(⊖) is a counter anion. In some embodiments, X^(⊖) is halide,phosphate, sulfate, trifluoroacetate, toluenesulfonate, acetate,formate, citrate, ascorbate, mesylate (methanesulfonate), or benzoate.

In some embodiments, a compound of formula (II) comprises the formula:

or a salt, free base, or combination thereof.

In some embodiments, at least one R² is not hydrogen, optionally,wherein at least one R² is t-butyloxycarbonyl. In some embodiments, thecompound of formula (II) comprises the formula:

or a salt, free base, or combination thereof.

In some embodiments, a compound of formula (II) comprises the formula:

or a salt, free base, or combination thereof.

In some embodiments, m is 3. In some embodiments, m is an integerbetween 3 and 12, inclusive. In some embodiments, R³ is halide; andR⁴-R⁶ are hydrogen. In some embodiments, R³ is Br. In some embodiments,R¹ is C₁-C₆ alkyl, haloalkyl, or aryl. In some embodiments, R¹ ismethyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, orhexyl. In some embodiments, R¹ is haloalkyl. In some embodiments, R¹ isCF₃. In some embodiments, R¹ is phenyl (Ph), optionally substituted. Insome embodiments, R¹ is 4-CH₃C₆H₄, 2,4,6-(CH₃)₃C₆H₂, or C₆H₄X, wherein Xis halide. In some embodiments, n is 1. In some embodiments, m is aninteger between 1 and 10 inclusive, or between 1 and 8 inclusive, orbetween 1 and 6 inclusive. In some embodiments, F is isotopicallyenriched with ¹⁸F.

In one embodiment, a compound of formula (II) comprises the formula:

In one aspect, a compound of formula (II) comprises the formula

In one aspect, a compound of formula (II) comprises the formula:

In one aspect, a compound of formula (II) comprises the formula:

In some embodiments, a compound of formula (II) comprises the formula:

In some embodiments, the compound comprising formula (I), formula (II),and/or formula (IV) is provided as a solution in a solvent.

In some embodiments, the conditions suitable for deprotection compriseexposing the compound of formula (I) and/or formula (II) to an acid orto an acidic environment. In some embodiments, the acid is hydrochloricacid, formic acid, sulfuric acid, benzoic acid, acetic acid,trifluoroacetic acid, p-toluenesulfonic acid, phosphoric acid, ormethanesulfonic acid. An acidic environment may be, for example, a pHequal to or less than 4, equal to or less than 3, equal to or less than2, or equal to or less than 1.

In some embodiments, the suitable conditions comprise reacting at orabove room temperature. In some embodiments, conditions suitable fordeprotection and/or fluorination may comprise a temperature ranging fromabout 100° C. to about 150° C., including a temperature of about 100° C.

In some embodiments, the suitable conditions comprise reacting at atemperature of about 50° C., or about 60° C., or about 70° C., or about80° C., or about 90° C., or about 100° C., or about 110° C., or about120° C., or about 150° C., or about 170° C., or about 200° C., or about225° C., or about 250° C. for a period of about 5 minutes or less, orabout 10 minutes or less, or about 20 minutes or less, or about 30minutes or less.

In some embodiments, the suitable conditions comprise a solution pH ofequal to or less than about 13, or equal to or less than about 12, orequal to or less than about 11. In some embodiments, the suitableconditions comprise a solution pH of between about 8 and about 9, orbetween about 8 and about 10, or between about 7 and about 8. In someembodiments, conditions suitable for fluorination comprise a pH in therange of about 8-13, about 9-13, about 10-13, or about 10-12.

In some embodiments, the solvent is benzene, toluene, xylene, diethylether, glycol, diethyl ether, hexane, pentane, methylene chloride,chloroform, dioxane, tetrahydrofuran, ethyl acetate, water, or mixturesthereof. In some embodiments, the compound comprising formula (V) isisolated using column chromatography.

In some embodiments, the step of reacting comprises exposing a compoundcomprising formula (IV) to a source of fluoride. In some embodiments,the source of fluoride is isotopically enriched with ¹⁸F. In someembodiments, the source of fluoride is NaF or KF.

In some embodiments, the suitable conditions further comprise exposing acompound comprising Formula (II) or Formula (IV) to a source of fluoridein the presence of an ammonium salt or a bicarbonate salt. In someembodiments, the molar ratio of ammonium salt or bicarbonate salt to thecompound of formula (IV) is less than or equal to about 10:1, or lessthan or equal to about 9:1, or less than or equal to about 8:1, or lessthan or equal to about 7:1 or less than or equal to about 6:1, or lessthan or equal to about 5:1, or less than or equal to about 4:1, or lessthan or equal to about 3:1, or less than or equal to about 2:1, or lessthan or equal to about 1:1. In some embodiments, the ammonium salt is anammonium bicarbonate salt, ammonium hydroxide salt, ammonium acetatesalt, ammonium lactate salt, ammonium trifluoroacetate salt, ammoniummethanesulfonate salt, ammonium p-toluenesulfonate salt, ammoniumnitrate salt, ammonium iodide salt, or ammonium bisulfate salt. In someembodiments, the bicarbonate salt is a tetraalkylammonium bicarbonate.In some embodiments, the ammonium salt or the bicarbonate salt comprisesthe formula:

R₄NHCO₃,

wherein R₄ is alkyl. In some embodiments, the reacting is carried out inthe presence of a cryptand.

In embodiments, a method comprises reacting a compound comprisingformula (XI):

or a salt, free base, or combination thereof, under conditions suitableto form a compound comprising formula (II):

or a salt, free base, or combination thereof, wherein R¹ is alkyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,alkenyl, alkynyl, heterocyclyl, or haloalkyl, each optionallysubstituted; each R² can be the same or different and is hydrogen or anitrogen-protecting group, provided at least one R² is not hydrogen; R³,R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted; each R⁷ can be the same ordifferent and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl,aryl, heteroaryl, or heterocyclyl, each optionally substituted; each R⁸can be the same or different and is hydrogen, alkyl, heteroalkyl,cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, —OH, alkoxy,—NH₂, alkylamino, —SH, or alkylthiol, each optionally substituted; m isan integer between 1 and 12, inclusive; and n is an integer between 1and 4, inclusive.

In yet another aspect, the invention provides particular salts ofimaging agents and/or their precursors. In one embodiment, a saltcomprises formula (VI):

wherein X^(⊖) is formate.

In another embodiment, a salt comprises formula (VII):

wherein X^(⊖) is ascorbate.

In some embodiments, the salt is a citrate salt or a trifluoroacetatesalt comprising the cation of formula (VI) or (VII).

In some embodiments, the fluorine of a salt is isotopically enrichedwith ¹⁸F.

In some embodiments, a pharmaceutically acceptable compositioncomprising a salt as described herein and optionally a pharmaceuticallyacceptable excipient is provided.

In some embodiments, a kit is provided comprising a salt or compositionas described herein and instructions for use.

In another aspect, methods of imaging are provided. In one embodiment, amethod of imaging a subject comprises administering a dose of apharmaceutically acceptable composition comprising an imaging agent,including salts thereof, as described herein, wherein the fluorine isisotopically enriched with ¹⁸F, and optionally a pharmaceuticallyacceptable excipient, to a subject; and acquiring at least one image ofa portion of the subject. In some embodiments, the maximum dose of theimaging agent is approximately 15 mCi or less, 14 mCi or less, 13 mCi orless, 12 mCi or less, 11 mCi or less or 10 mCi or less.

In one aspect, the invention provides use of a salt as described hereinfor imaging a portion of a subject.

In some embodiments, a method of imaging a subject is provided thatcomprises administering a dose of a compound comprising the formula:

or a free base, pharmaceutically acceptable salt, or combinationthereof, to a subject, wherein the maximum dose of the compoundadministered to the subject is approximately 15 mCi or less; andacquiring at least one image of a portion of the subject.

In some embodiments, a method for detecting norepinephrine transporter(NET) in a portion of a subject is provided, the method comprisingadministering a dose of a compound comprising the formula:

or a free base, pharmaceutically acceptable salt, or combinationthereof, to a subject, wherein the maximum dose of the compoundadministered to the subject is less than approximately 14 mCi; andacquiring at least one image of the portion of the subject, wherein theimage detects NET in the subject.

In some embodiments, the maximum dose of the compound administered tothe subject is approximately 13 mCi or less, is between approximately 10mCi and approximately 13 mCi, or is between approximately 8 mCi andapproximately 10 mCi.

In some embodiments, the step of acquiring employs positron emissiontomography. In some embodiment, the portion of the subject being imagedis at least a portion of the cardiovascular system, the heart, or is atleast a portion of the heart.

In some embodiments, the method further comprises determining thepresence or absence of a cardiovascular disease or condition in thesubject.

In some embodiments, the compound is provided for administration in asolution comprising between approximately 1% and approximately 10%ethanol and between approximately 25 mg/mL and approximately 75 mg/mlascorbic acid.

In some embodiments, the method further comprises administering a seconddose of the compound to the subject at a time subsequent to the firstdose; and acquiring at least one image of the portion of the subjectafter the administration of the second dose of the compound. In someembodiments, the method further comprises comparing the at least oneimage acquired after the first dose with the at least one image acquiredafter the second dose; and determining the presence or absence ofdifferences between the cardiac sympathetic innervation at the time ofadministration of the first and second dose of the compound to thesubject.

In some embodiments, presence of NET indicates presence of a condition.In some embodiments, the condition is a tumor.

In some embodiments, the detecting comprises determining level, density,localization, and/or function of NET in the portion of the subject.

In some embodiments, the method further comprises assessing cardiacsympathetic innervation in the subject.

In some embodiments, the step of determining comprises determininglevel, density, localization, or function of NETs in the portion of thesubject.

In some embodiments, image data from dynamic images are used todistinguish changes in local or global blood flow from changes in localor global NET function or distribution.

In some embodiments, the method further comprises providing image datausing another imaging agent, and determining blood flow based on theimage data to distinguish local or global blood flow from local orglobal changes in NET function or distribution.

In some embodiments, the method further comprising assessing cardiacsympathetic innervation in the subject.

In some embodiments, at least a portion of the compound is present as apharmaceutically acceptable salt. In some embodiments, the salt is aformate salt or the ascorbate salt of the compound. In some embodiments,the salt is the citrate salt or the trifluoroacetate salt of thecompound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a nucleophilic [¹⁸F]-fluorination reactionusing an imaging agent precursor and a fluoride source to form animaging agent of the invention.

FIG. 2 shows a flow chart showing an exemplary method for synthesizingan imaging agent of the invention.

FIG. 3 and FIG. 4 are schematic representations of exemplary cassetteswith associated columns and reagents for synthesizing an imaging agentof the invention using a modified GE TRACERLab-MX chemistry module.

FIG. 5 is a schematic representation of a system for synthesizing animaging agent of the invention using a modified Explora GN chemistrymodule.

FIG. 6 shows an exemplary synthesis of an imaging agent precursor of theinvention.

FIG. 7 show graphs of weight percent versus time for the sulfuric acidsalt of imaging agent precursor-1 and the trifluoroacetic acid salt ofimaging agent precursor-1.

FIG. 8A-FIG. 8F show HPLC chromatograms for compounds synthesizedaccording to methods described herein.

FIG. 9A shows a graph illustrating the changes in product distributionas a function of carbonate stoichiometry.

FIG. 9B shows various side products which may be formed during thesynthesis of imaging agent-1 from imaging agent precursor-1.

FIG. 9C shows a graph illustrating the changes in product distributionof imaging agent-1 as a function of Et₄NHCO₃ stoichiometry.

FIG. 10 shows a graph illustrating the tissue distribution of imagingagent-1 in tumor-bearing mice.

Other aspects, embodiments, and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to compounds, compositionsthereof, systems comprising such compounds, reagents, cassettes,methods, kits, and apparatuses for the synthesis and/or use of imagingagents and precursors thereof. In some aspects, the invention generallyrelates to an imaging agent of the invention (i.e., an imaging agent ofFormula (I), including an imaging agent of formula (V), such as imagingagent-1) synthesized using methods described herein. The imaging agentsof the invention may be used to image an area of interest in a subject,including, but not limited to, the heart, a portion of the heart, thecardiovascular system, cardiac vessels, brain, and other organs.

In some embodiments, the present invention provides methods forsynthesizing an imaging agent precursor of the invention that can bereacted with an imaging moiety (or a source thereof) to form an imagingagent. It is advantageous to utilize methods which involve high-yieldingreactions and a relatively low number of synthetic, purification, and/orformulation events in the preparation of an imaging agent precursorand/or imaging agent. Accordingly, many of the methods provided hereinfor synthesizing an imaging agent precursor and/or imaging agent producethe compounds in fewer steps than previously reported, with greater easeof synthesis, and/or with higher yield. In certain embodiments, thefluorination of an imaging agent precursor comprising a sulfonateleaving group is performed with a fully deprotected form of theprecursor eliminating the need for a subsequent deprotection step.Therefore, the last synthetic step is the fluorination reactioneliminating the loss of isotopically labeled material in subsequentsteps.

The methods and compositions of this disclosure provide variousadvantages over the methods, compounds, and compositions known in theart. As another example, some of the compounds provided herein are saltsassociated with a counter anion, wherein the counter anion has beenunexpectedly found to improve the solubility, yield, stability, and/orease of purification of the compound. For example, the counter anion insome instances influences numerous aspects of the manufacture of animaging agent, or precursor thereof, or a composition thereof, including(1) solubility of the imaging agent precursor and/or imaging agent, (2)purity of the imaging agent precursor and/or imaging agent, and (3)stability of the imaging precursor and/or imaging agent.

Imaging Agents

In some aspects, imaging agents for imaging an area of interest of asubject are provided. In certain embodiments, the imaging agent islabeled with ¹⁸F and is useful in PET imaging. In some embodiments, theimaging agent is a compound comprising formula (I):

or a salt, free base, or combinations thereof, wherein:

R³, R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted;

each R² can be the same or different and is hydrogen or anitrogen-protecting group;

each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, or heteroaryl,each optionally substituted;

each R⁸ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, —OH,alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, each optionallysubstituted;

m is an integer between 1 and 12, inclusive; and

n is an integer between 1 and 4, inclusive.

In certain embodiments, the imaging agent is a compound comprisingformula (V):

or a salt, free base, or combinations thereof, wherein R³, R⁴, R⁵, R⁶,m, and n are as defined above.

In certain embodiments, the compound of formula (I) comprises formula:

wherein at least one R² is a nitrogen protecting group. In certainembodiments, the nitrogen protecting group is a Boc protecting group. Incertain embodiments, one, two, or three R² groups are nitrogenprotecting groups (e.g., Boc protecting groups), and the other R² groupsare hydrogen. In certain embodiments, the fluorine of the compounds isisotopically enriched with ¹⁸F. Fully protected, partially protected,and fully unprotected forms of compounds comprising formula (I)isotopically enriched with ¹⁸F may be useful as imaging agents.

A non-limiting example of an imaging agent, referred to herein asimaging agent-1, comprises the formula:

As used herein, the term imaging agent-1 may also refer to a salt and/ora free base, or combinations thereof, of the above compound, such as aformate salt (Formula (VI)), an ascorbate salt (Formula (VII)), acitrate salt (Formula (IX)), or a trifluoroacetic acid salt (Formula(X)), as described herein.

For the sake of convenience and brevity, various aspects and embodimentsof the invention are described in terms of imaging agent-1. However, itis to be understood that, unless otherwise specified, the inventioncontemplates the synthesis and use of imaging agents other than imagingagent-1 in these various aspects and embodiments. Such imaging agentsmay be compounds of formula (I) and/or compounds of formula (V), asdescribed herein.

As used herein, the term “imaging agent” refers to any chemical compoundthat includes an imaging moiety. An “imaging moiety” refers to an atomor group of atoms that is capable of producing a detectable signalitself, or upon exposure to an external source of energy (e.g.,electromagnetic radiation, ultrasound, and the like). Nuclear medicineimaging agents may comprise radioisotopes as the imaging moiety. Forexample, nuclear medicine imaging agents can include ¹¹C, ¹³N, ¹⁸F,⁷⁶Br, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga,and ⁶⁸Ga as the imaging moiety. In some embodiments, the imaging moietyis ¹⁸F. Imaging agents comprising ¹⁸F have been used for imaging hypoxiaand cancer (Drugs of the Future 2002, 27, 655-667).

Imaging agents allow for the detection, imaging, and/or monitoring ofthe presence and/or progression of a condition, pathological disorder,and/or disease. Typically, the imaging agent may be administered to asubject in order to provide information relating to at least a portionof the subject (e.g., human). In some cases, an imaging agent may beused to highlight a specific area of a subject, rendering organs, bloodvessels, tissues, and/or other portions more detectable and more clearlyimaged. By increasing the detectability and/or image quality of the areabeing studied, the presence and extent of disease and/or injury can bedetermined.

In some embodiments, an imaging agent comprising an isotope such as aradioisotope may be referred to as being “isotopically enriched.” An“isotopically enriched” composition refers to a composition comprising apercentage of one or more isotopes of an element that is more than thepercentage (of such isotope) that occurs naturally. As an example, acomposition that is isotopically enriched with a fluoride species may be“isotopically enriched” with fluorine-18 (¹⁸F). Thus, with regard to aplurality of compounds, when a particular atomic position is designatedas ¹⁸F, it is to be understood that the abundance (or frequency) of ¹⁸Fat that position (in the plurality) is greater, including substantiallygreater, than the natural abundance (or frequency) of ¹⁸F, which isessentially zero. In some embodiments, a fluorine designated as ¹⁸F mayhave a minimum isotopic enrichment factor of about 0.001% (i.e., about 1out of 10⁵ fluorine species is ¹⁸F), 0.002%, 0.003%, 0.004%, 0.005%,0.006%, 0.007%, 0.008%, 0.009%, 0.01%, about 0.05%, about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.75%, about 1%, about2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or greater. The minimum isotopic enrichment factor, in someinstances, may range from about 0.001% to about 1%. The isotopicenrichment of the compounds provided herein can be determined usingconventional analytical methods known to one of ordinary skill in theart, including mass spectrometry and HPLC.

In some embodiments, methods and systems of this disclosure use orcomprise compounds of formula (I) or (V), including, without limitation,imaging agent-1. In some embodiments, the present invention relates tomethods of imaging, including methods of imaging in a subject thatincludes administering a composition or formulation that includes animaging agent (e.g., an imaging agent comprising formula (I) or formula(V), such as imaging agent-1) to the subject by injection, infusion, orany other known method, and imaging a region of interest of the subject.Regions of interest may include, but are not limited to, the heart, aportion of the heart, cardiovascular system, cardiac vessels, pancreas,adrenal glands, salivary glands, thymus, or other organs with highsympathetic innervation or high imaging agent uptake. Regions ofinterest may also include tumors. In certain embodiments, the imagingagent is used as a radiotracer for mapping the cardiac nerve terminal invivo using positron emission tomography (PET) or other imagingtechniques. An event of interest can be imaged and detected and/or otherinformation may be determined using methods and/or systems of thedisclosure.

The imaging agents of the invention, including imaging agent-1, may actas norepinephrine transporter ligands that target or bind NET. In someembodiments, the methods comprise detecting MET, including determiningNET levels, in a subject, wherein determining may comprise determiningthe level, density, function, and/or localization of NET in a subject.In certain embodiments, without wishing to be bound by a particulartheory, the imaging agent binds to norepinephrine transporters (NET)allowing for imaging of cardiac sympathetic innervation or activity.Accordingly, in some aspects, methods for assessing cardiac sympatheticinnervation and/or myocardial sympathetic function are provided.

Imaging Agent Precursors

In other aspects, imaging agent precursors useful in the preparation ofimaging agents of the invention are provided. An exemplary synthesis ofimaging agent precursor 1 is shown in FIG. 6. In certain embodiments, animaging agent precursor of the invention comprises a leaving group(e.g., a sulfonate) that can be substituted with a nucleophile in asubstitution reaction. The imaging agent precursor may also includevarious functional groups that are optionally protected. Earlierprecursors in the synthesis of imaging agents of the invention are alsoencompassed by the present invention.

In certain embodiments, the present invention provides a compound (e.g.,an imaging agent precursor) comprising formula (II):

or a salt, free base, or combinations thereof, wherein

R¹ is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,arylalkyl, heteroarylalkyl, alkenyl, alkynyl, or haloalkyl, eachoptionally substituted;

R³, R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted;

each R² can be the same or different and is hydrogen or anitrogen-protecting group;

each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, or heteroaryl,each optionally substituted;

each R⁸ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, heteroaryl, —OH,alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, each optionallysubstituted;

m is an integer between 1 and 12, inclusive; and

n is an integer between 1 and 4, inclusive. In some embodiments, acompound of formula (II) is an imaging agent precursor.

In certain embodiments, the imaging agent precursor is a compoundcomprising Formula (IV):

or a salt, free base, or combination thereof, wherein R¹, R³-R⁶, m, andn are as defined herein.

A non-limiting example of an imaging agent precursor, referred to hereinas imaging agent precursor-1, comprises the formula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor, referred toherein as imaging agent precursor-2, comprises the formula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor comprises theformula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor comprises theformula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor comprises theformula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor comprises theformula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor comprises theformula:

or a salt, free base, or combinations thereof.

Another non-limiting example of an imaging agent precursor comprises theformula:

or a salt, free base, or combinations thereof.

For the sake of convenience and brevity, various aspects and embodimentsof the invention are described in terms of imaging agent precursor-1and/or imaging agent precursor-2. However, it is to be understood that,unless otherwise specified, the invention contemplates the synthesis anduse of imaging agent precursors other than imaging agent precursor-1 and-2 in these various aspects and embodiments. Such imaging agentprecursors may be compounds of Formula (II) and/or compounds of Formula(IV) and/or compounds of Formula (III), as described herein.

In certain embodiments, a salt of a compound of formula (II) isprovided. That is, a compound of formula (II) may be charged and may beassociated with a counter ion. In some cases, the compound of formula(II) is positively charged. In a particular embodiment, the guanidinefunctional group of the compound of formula (II) is protonated andtherefore positively charged such that a salt of a compound of formula(II) comprises formula (III):

wherein X^(⊖) is a counter anion. As will be understood by those ofordinary skill in the art, in embodiments described herein wherein acompound comprises a compound of formula (II), or a variation thereof,the compound may be present, at least in part, in a salt form. Forexample, any compound described herein comprising a neutral and/orunprotonated guanidine functional group may also be present as aprotonated guanidine functional group (e.g., associated with a counteranion).

Those of ordinary skill in the art will be aware of suitable counteranions. In addition, those of ordinary skill in the art will be awarethat the counter anion X^(⊖) may have a charge of greater than (−1)(e.g., (−2), (−3)), and in such embodiments, each counter anion X^(⊖)may be associated with more than one molecule of a compound of thepresent invention. Non-limiting examples of suitable counter anionsinclude the conjugate base of inorganic acids (e.g., chloride, bromide,iodide, fluoride, nitrate, sulfate, phosphate) or from the conjugatebase of organic acids (e.g., carboxylate, acetate, benzoate, tartrate,adipate, lactate, formate, maleate, glutamate, ascorbate, citrate,gluconate, oxalate, succinate, pamoate, salicylate, isethionate,succinamate, mono-diglycollate, di-isobutyrate, glucoheptonate). Stillother non-limiting examples of salts include adipate, alginate,aminosalicylate, anhydromethylenecitrate, arecoline, aspartate,bisulfate, camphorate, digluconate, dihydrobromide, disuccinate,glycerophosphate, hemisulfate, fluoride, iodide,methylenebis(salicylate), napadisylate, oxalate, pectinate, persulfate,phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate,undecanoate, acetate, benzenesulfonate, benzoate, bicarbonate,bitartrate, bromide, calcium edentate, camyslate, carbonate, chloride,citrate, dihydrochloride, edentate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, bromide, chloride, hydroxynaphthoate,iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate,mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, teoclate, andtriethiodide (see Berge et al., Journal of Pharmaceutical Sciences,66(1), 1977, 1-19). In certain embodiments, the salt is a mesylate(i.e., methanesulfonate), phosphate, sulfate, acetate, formate,benzoate, trifluoroacetate, or tosylate salt of a compound of formula(II). In certain embodiments, the salt is a mesylate (i.e.,methanesulfonate), acetate, formate, benzoate, trifluoroacetate, ortosylate salt of a compound of formula (II).

In some embodiments, R¹ is alkyl, haloalkyl, or aryl. In some cases, R¹is alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,t-butyl, pentyl, hexyl). In some cases, R¹ is haloalkyl (e.g., —CF₃,—CHF₂, —CH₂F, —CF₂CF₃, —CH₂CF₃). In some cases, R¹ is aryl, optionallysubstituted. In certain embodiments, R¹ is substituted or unsubstitutedphenyl. In certain embodiments, R¹ is unsubstituted phenyl. In somecases, R¹ is substituted phenyl (e.g., 4-CH₃Ph, 2,4,6-(CH₃)₃C₆H₂, C₆H₄Xwherein X is halide (e.g., 4-BrC₆H₄)).

In some embodiments n is an integer between 1 and 4, inclusive, or is 1,2, 3, or 4.

In some embodiments, m is an integer between 1 and 12, inclusive; or 1and 10, inclusive; or 1 and 8, inclusive; or 1 and 6, inclusive; or is1, 2, 3, 4, 5, or 6. In some embodiments, m is an integer between 3 and12, inclusive.

As described above, R² may be a nitrogen protecting group.Nitrogen-protecting groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference. For example, nitrogen protectinggroups include, but are not limited to, carbamates (including methyl,ethyl and substituted ethyl carbamates (e.g. Troc), to name a few),amides, cyclic imide derivatives, N-alkyl and N-aryl amines, iminederivatives, and enamine derivatives, to name a few. In someembodiments, the nitrogen-protecting group is carbobenzyloxy (Cbz),p-methoxybenzyl carbonyl (MeOZ), t-butyloxycarbonyl (Boc),9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl(Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl(PMP), or p-toluenesulfonyloxy (Ts). In certain embodiments, at leastone R² is t-butyloxycarbonyl (Boc).

Nitrogen-protecting groups such as amide groups include, but are notlimited to, formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen-protecting groups such as carbamate groups include, but are notlimited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethylcarbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate,9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen-protecting groups such as sulfonamide groups include, but arenot limited to, p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), J3-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen-protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In some embodiments, R⁴, R⁵, and R⁶ are hydrogen; and R³ is C₁-C₆ alkyl,hetero-C₁-C₆ alkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or —C(═O)R⁸, eachoptionally substituted. In some cases, R³ is halo (e.g., F, Cl, Br, I).In certain embodiments, R³ is bromo. In a particular embodiment, R⁴, R⁵,and R⁶ are hydrogen; and R³ is bromo, for example, such that thecompound of formula (II) comprises the structure:

In some embodiments, each R² is hydrogen such that the compound offormula (II) comprises the structure:

In a particular embodiment, R⁴, R⁵, and R⁶ are hydrogen; R³ is bromo;and each R² is hydrogen, for example, such that the compound of formula(II) has the structure:

In other embodiments, at least one R² is not hydrogen. For example, thecompound of formula (II) may be one of the formulae:

As described herein, these compounds may be present, as a salt, freebase, or combination thereof.

In some embodiments, m is 3, n is 1, R³ is Br (or another halogen), andR⁴, R⁵, and R⁶ are all H, such that a compound of formula (II) comprisesthe structure:

wherein each of R¹ and R² are as defined above and described inembodiments herein, both singly and in combination. Further, in somecases, each R² is H, such that the compound of formula (II) comprisesthe structure:

wherein R¹ is as defined above and described in embodiments herein.

In certain embodiments, a compound of formula (II) comprises thestructure:

or a salt, free base, or combination thereof.

In certain embodiments, the present invention provides compounds usefulin the synthesis of compounds of Formula (II). In certain embodiments,the present invention provides a compound of formula:

or a salt, free base, or combination thereof; wherein each R² can be thesame or different and is hydrogen or a nitrogen-protecting group; and mis an integer between 3 and 12, inclusive. In one embodiment, m is 3.

In certain embodiments, the invention provides a compound comprisingformula:

or a salt, free base, or combination thereof.

In certain embodiments, the invention provides a compound comprisingformula:

or a salt, free base, or combination thereof.

In one embodiment, the invention provides a compound comprising formula:

or a salt, free base, or combination thereof.

In one embodiment, the invention provides a compound comprising formula:

or a salt, free base, or combinations thereof.

In certain embodiments, m is an integer between 3 and 10, inclusive;between 3 and 6, inclusive; or between 3 and 5, inclusive. In certainembodiments, m is 3, 4, 5, or 6. In certain embodiments, m is 3.

In some embodiments, all R² are hydrogen. In other embodiments, at leastone R² is a nitrogen protecting group (e.g., nitrogen protecting groupsdescribed herein). In other embodiments, at least two R² are nitrogenprotecting groups (e.g., nitrogen protecting groups described herein).In other embodiments, at least three R² are nitrogen protecting groups(e.g., nitrogen protecting groups described herein). In someembodiments, the nitrogen-protecting group is carbobenzyloxy (Cbz),p-methoxybenzyl carbonyl (MeOZ), t-butyloxycarbonyl (Boc),9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl(Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl(PMP), or p-toluenesulfonyloxy (Ts). In certain embodiments, at leastone R² is t-butyloxycarbonyl (Boc). In certain embodiments, at least twoR² are t-butyloxycarbonyl (Boc).

In another aspect, the invention provides a compound comprising formula:

or a salt, free base, or combination thereof, wherein m is an integerbetween 3 and 12, inclusive. In certain embodiments, m is an integerbetween 3 and 10, inclusive; between 3 and 6, inclusive; or between 3and 5, inclusive. In certain embodiments, m is 3, 4, 5, or 6. In certainembodiments, m is 3.

In one embodiment, the invention provides a compound comprising formula:

or a free base, salt, or combination thereof.

In one aspect, the invention provides a compound comprising formula:

or a salt thereof, wherein m is an integer between 3 and 12, inclusive.In certain embodiments, m is an integer between 3 and 10, inclusive;between 3 and 6, inclusive; or between 3 and 5, inclusive. In certainembodiments, m is 3, 4, 5, or 6. In certain embodiments, m is 3.

In one embodiment, the invention provides a compound comprising formula:

Methods of Synthesizing Imaging Agent Precursors

In other aspects, methods of synthesizing imaging agent precursors ofthe invention and imaging agents of the invention are provided. Incertain embodiments, an imaging agent precursor with a leaving group(e.g., sulfonate) is reacted with a nucleophile in a substitutionreaction to yield an imaging agent of the invention, or a protected formthereof. Synthetic methods are also provided for preparing earlierprecursors in the synthesis of imaging agents of the invention, forexample, synthetic methods exemplary steps of which are shown in FIG. 6.

In some embodiments, the present invention provides methods forsynthesizing imaging agent precursors of the invention. The methodsdescribed herein may be used for the synthesis of a variety of imagingagent precursors. Generally, the imaging agent precursor includes aleaving group that is replaced by an imaging moiety, such as an ¹⁸Fspecies.

The imaging agent precursors of the invention (e.g., compounds ofFormula (II)) may be prepared in a variety of different ways. In certainembodiments, the free hydroxyl group of an alcohol that comprisesformula (XI):

or a salt, free base, or combinations thereof, wherein

R³, R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted;

each R² can be the same or different and is hydrogen or anitrogen-protecting group;

each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, or heteroaryl,each optionally substituted;

each R⁸ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, heteroaryl, —OH,alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, each optionallysubstituted;

m is an integer between 1 and 12, inclusive; and

n is an integer between 1 and 4, inclusive,

is converted into a suitable leaving group (e.g., a sulfonate leavinggroup) to yield a compound comprising Formula (II). Each of R²-R⁸, m,and n are as defined above and described in embodiments herein, bothsingly and in combination, unless stated otherwise. Sulfonate leavinggroup methodology is reviewed in Netscher, Recent Res. Dev. Org. Chem.7:71-83, 2003, which is incorporated herein by reference. In certainembodiments, the free hydroxyl group is converted into a tosylate(4-methylbenzenesulfonate) using tosyl halide (e.g., tosyl chloride). Incertain embodiments, the free hydroxyl group is converted into abesylate (benzenesulfonate) using a besylate halide (e.g., besylatechloride). In certain embodiments, the free hydroxyl group is convertedinto a nosylate (4-nitrobenzenesulfonate) using a nosylate halide (e.g.,nosylate chloride). In other embodiments, the free hydroxyl group isconverted into bromobenzenesulfonate using a bromobenzenesulfonatehalide (e.g., bromobenzenesulfonate chloride). In other embodiments, thefree hydroxyl group is converted into a mesylate (methanesulfonate)using a mesyl halide (e.g., mesyl chloride). In other embodiments, thefree hydroxyl group is converted into a triflate(trifluoromethanesulfonate) using triflic anhydride or a triflic halide.As would be appreciate by one of skill in the art, other sulfonates maybe used in the imaging agent precursors of the invention. Typically thepreparation of the sulfonate comprising Formula (II) is performed in anaprotic solvent (e.g., dichloromethane, THF) at or around roomtemperature in the presence of a base such a DMAP and/or atrialkylamine.

The alcohol comprising formula (XI):

may be prepared based on synthetic methodologies disclosed in PCTPublication No. WO 2008/083056, which is incorporated herein byreferenced. Furthermore, exemplary syntheses of various imaging agentprecursors of Formula (II), including salt forms thereof, are providedin Examples 1-13 and FIG. 6.

In certain embodiments, the alcohol comprising formula (XI) is preparedby reacting a compound comprising formula:

or a salt, free base, or combination thereof, with a compound offormula:

wherein LG is a suitable leaving group. In one embodiment, m is 3.

In certain embodiments, the compound comprising formula:

is of formula:

In one embodiment, the compound comprising formula:

is of formula:

In one embodiment, the compound comprising formula:

is of formula:

In another aspect, the invention provides a method of preparing thestarting material for the previous reaction by reducing a compoundcomprising formula:

or a salt thereof, wherein m is an integer between 3 and 12, inclusive,with a reductant under suitable conditions to form a compoundcomprising:

or a salt, free base, or combination thereof. In one embodiment, m is 3.Exemplary agents useful in reducing a nitrile group (—CN) to a primaryamino group (—CH₂NH₂), include, but are not limited to, LiAlH₄ (LAH);hydrogen gas (H₂) in the presence of a metal catalyst (e.g., Pd, Pt,Ni); NaBH₄ and a transition metal salt to form the metal borate in situ(e.g., NiCl₂ to form nickel borate (NiBH₄) in situ; ZnCl₂ to form thezinc borate (ZnBH₄) in situ); NaBH₄ plus I₂; NaBH₄ plus H₂SO₄; NiBH₄;ZnBH₄; LiBH₄; and borane (e.g., BH₃/THF, BH₃/DCM). In one embodiment,the reductant is borane (e.g., BH₃/THF).

In some embodiments, the invention provides a method of deprotecting aguanidine functional group of a compound comprising formula (II):

For example, in some embodiments, a method comprises deprotecting aguanidine functional group of a compound comprising formula (II):

or a salt, free base, or combination thereof, under conditions suitableto form a compound comprising formula (IV):

or a salt, free base, or combination thereof, wherein

R¹ is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, alkenyl, alkynyl, heterocyclyl, or haloalkyl, eachoptionally substituted;

R³, R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted;

each R² can be the same or different and is hydrogen or anitrogen-protecting group;

each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl, or heterocyclyl,each optionally substituted;

each R⁸ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, —OH,alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, each optionallysubstituted;

m is an integer between 1 and 12, inclusive; and

n is an integer between 1 and 4, inclusive.

Each of R¹-R⁸, m, and n are as defined above and described inembodiments herein, both singly and in combination, unless statedotherwise.

Suitable conditions for deprotection of a guanidine functional group aredescribed herein. Such conditions may include an acidic environment(e.g., pH equal or less than 4, equal to or less than 3, equal to orless than 2, or equal to or less than 1). For example, in certainembodiments, one or more of R² is t-butyloxycarbonyl, and the step ofdeprotecting comprising treating a compound of formula (II) withtrifluoroacetic acid, hydrochloric acid, sulfuric acid, orp-toluenesulfonic acid. Such conditions for deprotection mayadditionally or alternatively include a temperature ranging from100-150° C.

The methods described herein may be carried out in any suitable solvent,including, but are not limited to, non-halogenated hydrocarbon solvents(e.g., pentane, hexane, heptane, cyclohexane), halogenated hydrocarbonsolvents (e.g., dichloromethane, chloroform, fluorobenzene,trifluoromethylbenzene), aromatic hydrocarbon solvents (e.g., toluene,benzene, xylene), ester solvents (e.g., ethyl acetate), ether solvents(e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane), andalcohol solvents (e.g., ethanol, methanol, propanol, isopropanol,tert-butanol). In certain embodiments, a protic solvent is used. Inother embodiments, an aprotic solvent is used. Non-limiting examples ofsolvents useful include acetone, acetic acid, formic acid, dimethylsulfoxide, dimethyl formamide, acetonitrile, p-cresol, glycol, petroleumether, carbon tetrachloride, hexamethyl-phosphoric triamide,triethylamine, picoline, and pyridine.

The methods may be carried out at any suitable temperature. In somecases, the method is carried out at about room temperature (e.g., about20° C., between about 20° C. and about 25° C., about 25° C., or thelike). In some cases, however, the method is carried out at atemperature below or above room temperature, for example, at about −78°C. at about −70° C., about −50° C., about −30° C., about −10° C., about−0° C., about 10° C., about 30° C., about 40° C., about 50° C., about60° C., about 70° C., about 80° C., about 90° C., about 100° C., about120° C., about 140° C., or the like. In some embodiments, the method iscarried out at temperatures above room temperature, for example, betweenabout 25° C. and about 120° C., or between about 25° C. and about 100°C., or between about 40° C. and about 120° C., or between about 80° C.and about 120° C. The temperature may be maintained by reflux of thesolution. In some cases, the method is carried out at temperaturesbetween about −78° C. and about 25° C., or between about 0° C. and about25° C.

The methods described herein may be carried out at any suitable pH, forexample, equal to or less than about 13, equal to or less than about 12,equal to or less than about 11, equal to or less than about 10, equal toor less than about 9, equal to or less than about 8, equal to or lessthan about 7, or equal to or less than about 6. In some cases, the pHmay be greater than or equal to 1, greater than or equal to 2, greaterthan or equal to 3, greater than or equal to 4, greater than or equal to5, greater than or equal to 6, greater than or equal to 7, or greaterthan or equal to 8. In some cases, the pH may be between about 2 andabout 12, or between about 3 and about 11, or between about 4 and about10, or between about 5 and about 9, or between about 6 and about 8, orabout 7.

The percent yield of a product may be greater than about 60%, greaterthan about 70%, greater than about 75%, greater than about 80%, greaterthan about 85%, greater than about 90%, greater than about 92%, greaterthan about 95%, greater than about 96%, greater than about 97%, greaterthan about 98%, greater than about 99%, or greater.

Methods of Synthesizing Imaging Agents

In other aspects, methods are provided for synthesizing imaging agents.The methods described herein may be used for the synthesis of a varietyof imaging agents of the invention from an imaging agent precursor ofthe invention.

Fluorination

In some cases, the imaging agent is formed by reacting an imaging agentprecursor (e.g., a compound comprising formula (II)-(IV)) with animaging moiety. The imaging agent precursor may include at least oneleaving group that is susceptible to being displaced by a nucleophilicimaging moiety, such as an ¹⁸F fluoride species. Thus, in certainembodiments, the method involves reacting an imaging agent precursorcomprising a leaving group with a source of an imaging moiety (e.g., afluoride species). For example, during the reaction, the imaging moietyreplaces the leaving group via a substitution reaction, such as anS_(N)2 or S_(N)1 reaction, thereby producing the imaging agent. Incertain embodiments, the fluorination reaction is a one-step procedurewhich does not require a subsequent deprotection step. That is, thefluorination step is performed on a fully deprotected imaging agentprecursor. A non-limiting example of a synthetic method for preparing animaging agent is shown in FIG. 1, wherein imaging agent precursor-1 isconverted into imaging agent-1. In some embodiments, multiplesubstitution reactions may occur through multiple leaving groups duringsynthesis of an imaging agent from an imaging agent precursor. Themethods described herein exhibit improved yields may allow for thesynthesis of imaging agents, including imaging agents comprising aradioisotope (e.g., ¹⁸F). The imaging agents may be useful as sensors,diagnostic tools, and the like. Synthetic methods for preparing animaging agent have also been designed to use an automated synthesissystem to prepare and purify imaging agents that comprise aradioisotope.

As described herein, in some cases, the method of synthesizing animaging agent of the invention may involve the use of one or morereagents (e.g., salts) that may facilitate a chemical reaction (e.g., asubstitution reaction). In certain embodiments, the choice of salt formmay allow for the fluorination of an unprotected imaging agentprecursor. Without wishing to be bound by a particular theory, thecounter anion may interact with the quanidine functional grouppreventing it from interfering with the fluorination reaction and/or mayprevent side reactions. In certain embodiments, the salt is a mesylate(i.e., methanesulfonate), phosphate, sulfate, acetate, formate,benzoate, trifluoroacetate, or tosylate salt of a compound of formula(II). In certain embodiments, the salt is a mesylate (i.e.,methanesulfonate), acetate, formate, benzoate, trifluoroacetate, ortosylate salt of a compound of formula (II).

In some embodiments, a method for synthesizing an imaging agentcomprises contacting an imaging agent precursor of the invention (e.g.,a compound comprising formula (II), (III), or (IV)) with a fluoridespecies resulting in the fluoride species replacing the leaving group ofthe precursor to produce an imaging agent (e.g., a compound comprisingformula (I)) comprising the fluorine species).

In certain embodiments, the method involves a nucleophilic fluorinationreaction. That is, an imaging agent precursor comprising a leaving groupis reacted in the presence of a fluoride species, whereby S_(N)2 orS_(N)1 displacement of the leaving group by the fluoride speciesproduces the imaging agent. In some embodiments, the fluoride species isisotopically enriched with ¹⁸F.

Those of ordinary skill in the art will be aware of suitable conditionsfor fluorinating a compound (e.g., a compound of formula (II), (III), or(IV)). For example, see International Patent Application No.PCT/US2011/024109, by Cesati, filed Feb. 8, 2011, herein incorporated byreference. In some cases, a compound of formula (II), (III), or (IV), ora salt, free base, or combination thereof, is exposed to a source offluorine, optionally enriched with an isotope of fluorine (e.g.,enriched with ¹⁸F). In some cases, the source of fluorine is a fluoridesalt (e.g., KF, NaF, tetralkylammonium fluoride).

The fluorine source may comprise or be associated with or may be used inconnection with another reagent. The reagent may be capable of enhancingthe reactivity of the fluorine species or otherwise facilitatingconversion of the precursor to the imaging agent. For example, in oneset of embodiments, the reagent may be used in combination with amultidentate ligand, such as a crown ether or a cryptand that is capableof chelating a metal ion. The multidentate ligand may be, for example,4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (i.e.,Kryptofix® 222). When the fluorine source is KF, cryptands having a highaffinity for potassium are useful as they chelate potassium and therebyincrease the reactivity of the fluoride ion. In some embodiments,cryptands having an affinity for potassium near that of Kryptofix® 222(e.g., 75%, 80%, 85%, 90%, 95%, or more of the Kryptofix® 222's affinityfor potassium) are used. The reaction conditions may comprise one ormore solvents.

In some embodiments, the fluorination occurs in the presence of K₂CO₃and Kryptofix® 222 (or any another cryptand having affinity for thecation of interest, including for example potassium, near that ofKryptofix® 222) in MeCN (acetonitrile) alone or in combination witht-BuOH, as the solvent. The molar ratio of K₂CO₃ to imaging agentprecursor (such as but not limited to imaging agent precursor-1 or -2)ranges from about 0.5:1 to about 5:1, more preferably 0.5:1 to 1:1. Insome embodiments, the molar ratio is about 0.66:1.

In some embodiments, the fluorination occurs in the presence oftetraalkylammonium carbonate or tetraalkylammonium bicarbonate in MeCNas the solvent. In some embodiments, the molar ratio oftetraalkylammonium carbonate or bicarbonate to imaging agent precursor(such as imaging agent precursor-1 or -2) is 5:1. In some embodiments,the molar ratio may range from about 7:1 to about 3:1, or from about 6:1to about 4:1, or about 5.5:1 to about 4.5:1. The tetraalkylammoniumcation may be tetraethylammonium or tetrabutylammonium but it is not solimited.

Compounds comprising formula (V):

or a salt, free base, or combination thereof, wherein each of R³-R⁶, m,and n are as defined above and described in embodiments herein, bothsingly and in combination, can be produced from a precursor using atwo-step or three-step process such as that described in InternationalPCT Publication WO 2008/083056 by Purohit et al., which is incorporatedherein by reference.

In contrast, the synthetic methods provided herein may involve asingle-step preparation of imaging agents of the invention (e.g.,compounds of formula (V), or a salt, free base, or combination thereof).The single-step method minimally involves fluorination of a completelyor partially deprotected precursor in the presence of, for example,K₂CO₃/Kryptofix® 222 (or other suitable alternatives to Kryptofix® 222)or tetraalkylammonium carbonate or bicarbonate, in MeCN alone or in anMeCN mixture (such as an MeCN and t-BuOH mixture). These methods areparticularly suitable when particular salt forms of the imaging agentprecursors of the invention are used. Such salts include halide,acetate, formate, citric, ascorbate, trifluoroacetate, toluenesulfonate,benzoate, acetate, phosphate, sulfate, tosylate, and mesylate.

In some cases, the methods further identify counter anions important inthe production of salts of a compound of formula (V). In some cases, thecounter anion may effect: (1) solubility of the precursor, (2) purity ofthe active pharmaceutical intermediate, and (3) stability of the drugproduct. In some cases, the trifluoroacetate anion was demonstrated tobe particularly effective. In certain embodiments, as described herein,the imaging agent precursor and/or the imaging agent is present in asalt form which aids in the reactivity and/or the stability of thereaction product and/or reactant during and/or after a deprotectionand/or fluorination reaction.

In some cases, the imaging agent precursor comprises a guanidinefunctional group which may or may not be deprotected prior to, or insome instances after, fluorination. For example, the guanidinefunctional group of a compound of formula (II) may or may not bedeprotected prior to fluorination. That is, in some cases, an imagingagent precursor comprising a protected guanidine functional group isfluorinated, optionally followed by deprotection. Alternatively, theguanidine functional group of an imaging agent precursor is deprotected(e.g., according to the methods described herein), followed byfluorination. As described herein, in certain embodiments, the fluorinesource is isotopically enriched with ¹⁸F.

In certain embodiments, a compound comprising formula (II) is firstfluorinated then deprotected. In certain embodiments, method comprisesfluorinating a compound comprising formula (II):

or a salt, free base, or combination thereof, under conditions suitableto form a compound comprising formula (I):

or a salt, free base, or combination thereof, wherein

R³, R⁴, R⁵, and R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted;

each R² can be the same or different and is hydrogen or anitrogen-protecting group;

each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl, or heterocyclyl,each optionally substituted;

each R⁸ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, —OH,alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, each optionallysubstituted;

m is an integer between 1 and 12, inclusive; and

n is an integer between 1 and 4, inclusive.

Each of R¹-R⁸, m, and n are as defined above and described inembodiments herein, both singly and in combination, unless otherwise.

Suitable conditions for fluorinating a compound are described herein.

In some instances, following fluorination of a compound comprisingformula (II) to form a compound comprising formula (I), the compoundcomprising formula (I) is deprotected completely or partially. Incertain embodiments, the method comprises deprotecting the compoundcomprising formula (I):

or a salt, free base, or combination thereof, provided at least one R²is not H, under conditions suitable to form a compound comprisingformula (V):

or a salt, free base, or combination thereof. Deprotection can occur,for example, under acidic conditions (e.g., pH equal to or less than 4),and optionally at elevated temperatures (e.g., ranging from about100-150° C.).

In some cases, however, an imaging agent precursor comprising adeprotected guanidine functional group is fluorinated. For example, incertain embodiments, the method comprises fluorinating a compoundcomprising formula (IV):

or a salt, free base, or combination thereof, under conditions suitableto form a compound of formula (V):

or a salt, free base, or combination thereof, wherein

R¹ is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, alkenyl, alkynyl, heterocyclyl, or haloalkyl, eachoptionally substituted;

R³, R⁴, R⁵, ad R⁶ can be the same or different and are individuallyhydrogen, C₁-C₆ alkyl, heteroalkyl, halide, —OR⁷, —SR⁷, —N(R⁷)₂, or—C(═O)R⁸, each optionally substituted;

each R⁷ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl, or heterocyclyl,each optionally substituted;

each R⁸ can be the same or different and is hydrogen, alkyl,heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, —OH,alkoxy, —NH₂, alkylamino, —SH, or alkylthiol, each optionallysubstituted;

m is an integer between 1 and 12, inclusive; and

n is an integer between 1 and 4, inclusive.

Each of R¹, R³-R⁸, m, and n are as defined above and described inembodiments herein, both singly and in combination, unless statedotherwise.

In some cases, it has been found, that the stability, solubility, and/orreactivity of a precursor sulfonic acid ester to a fluorinatedcounterpart is dependent on the derived guanidinium salt form. Forexample, an investigation of a series of mineral acid salts (e.g.,chloride, phosphate, and sulfate salts) demonstrated variable physicalproperties relevant to manufacture and long term storage capacity. Saltform development revealed solubility differences in multiple solventsystems relevant to modern fluorination chemistry including, forexample, MeCN, t-BuOH, and mixtures thereof. In some instances, agentprecursor solubility was correlated with overall fluorinationefficiency, as minimum imaging agent precursor concentration thresholdswere required in order to achieve preferential rates of fluorinationrelative to decomposition. In addition, in some cases, the reactionrates were also variable with selection of the counter anion, even atequivalent values of solution molarity.

In some embodiments, a method for synthesizing a fluorinated compoundcomprises reacting, in the presence of a reagent (e.g., a carbonate orbicarbonate ion), (i) a precursor of the fluorinated compound comprisinga substituent substituted with a halide or a sulfonate-containing group,with (ii) a salt comprising a fluoride species and weakly coordinatingcation.

As used herein, the term “leaving group” is given its ordinary meaningin the art of synthetic organic chemistry and refers to an atom or agroup capable of being displaced by a nucleophile. Examples of suitableleaving groups include, but are not limited to, halides (such aschloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy,alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy),arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, andhaloformates In some cases, the leaving group is a sulfonic acid ester,such as toluenesulfonate (tosylate, Ts), methanesulfonate (mesylate,Ms), p-bromobenzenesulfonyl (brosylate, Bs), ortrifluoromethanesulfonate (triflate, Tf). In some cases, the leavinggroup may be a brosylate, such as p-bromobenzenesulfonyl. In some cases,the leaving group may be a nosylate, such as 2-nitrobenzenesulfonyl. Theleaving group may also be a phosphineoxide (e.g., formed during aMitsunobu reaction) or an internal leaving group such as an epoxide orcyclic sulfate. In some embodiments, the leaving group is asulfonate-containing group. In some embodiments, the leaving group is atosylate group.

In some embodiments, one or more reagents is used in the reactionmixture comprising the imaging agent precursor and the fluoride species.A “reagent,” also referred to as an “additive,” is any chemical compoundadded to a reaction mixture. The reagent may be consumed or not consumedduring the reaction. The reagent may be a stoichiometric or catalyticreagent. Exemplary reagents include catalysts, salts, oxidants,reductants, chelating agents, bases, acids, metals, phase transferreagents, and others as would be appreciated by one of skill in the art.

The reagent may, in some cases, facilitate reaction between the imagingagent precursor and the fluoride species and/or may aid in stabilizingthe resultant imaging agent. For example, the fluoride species may haverelatively low reactivity (e.g., nucleophilicity), and addition ofcertain reagents may enhance the reactivity of the fluoride species. Asan illustrative embodiment, a fluorine species may be a negativelycharged fluoride ion (e.g., an isotopically enriched ¹⁸F ion), and areagent may be used to bind to any positively charged counter ionspresent within the reaction mixture, thereby enhancing the reactivity ofthe fluoride ion. An example of such a reagent is a cryptand such as,but not limited to, Kryptofix (e.g., Kryptofix®-222). In someembodiments, the reagent decreases the rate of undesired side reactions,as described below.

In some cases, the reagent may be combined with the fluoride speciesprior to its contact with the imaging agent precursor. For example, incertain embodiments a solution comprising the fluoride species and thereagent is prepared, and the solution is added to the imaging agentprecursor. In other embodiments, a solid comprising the fluoride speciesand the reagent is prepared, and the solid is contacted with the imagingagent precursor in solution. In certain embodiments, the fluoridespecies is adsorbed onto a solid support (e.g., an anion exchangecolumn), and a solution comprising the reagent is used to elute thefluoride species from the solid support. The eluted solution is thencontacted with the imaging agent precursor, or is concentrated toproduce a solid, which is then contacted with the imaging agentprecursor in solution.

In some embodiments, the reagent is a bicarbonate salt. As used herein,the term “bicarbonate salt” refers to a salt comprising a bicarbonate orhydrogen carbonate ion (HCO₃ ⁻ ion). The bicarbonate salt may be a metalbicarbonate, such as sodium bicarbonate, calcium bicarbonate, potassiumbicarbonate, and magnesium bicarbonate. In certain embodiments, thebicarbonate salt is potassium bicarbonate (KHCO₃). In some embodiments,the bicarbonate salt comprises a non-metal counter ion, such as ammoniumbicarbonate. For example, the bicarbonate salt may be atetraalkylammonium bicarbonate salt having the formula, R₄NHCO₃, whereinR₄ is alkyl. In some embodiments, R may be a lower alkyl, such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, or the like. In certainembodiments, the ammonium salt is Et₄NHCO₃. In other embodiments, thesalt is Me₄NHCO₃, i-Pr₄NHCO₃, n-Pr₄NHCO₃, n-Bu₄NHCO₃, i-Bu₄NHCO₃, ort-Bu₄NHCO₃.

In some embodiments, the reagent is a carbonate salt. As used herein,the term “carbonate salt” refers to a salt comprising a carbonate ion(CO₃ ⁻² ion). The carbonate salt may be a metal carbonate, such assodium carbonate, calcium carbonate, potassium carbonate, and magnesiumcarbonate. In certain embodiments, the carbonate salt is potassiumcarbonate (K₂CO₃). In some embodiments, the carbonate salt comprises anon-metal counter ion, such as ammonium carbonate. For example, thecarbonate salt may be a tetraalkylammonium carbonate salt having theformula, (R₄N)₂CO₃, wherein R is alkyl. In some embodiments, R may be alower alkyl, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, or thelike. In certain embodiments, the ammonium salt is (Et₄N)₂CO₃. In otherembodiments, the salt is (Me₄N)₂CO₃, (i-Pr₄N)₂CO₃, (n-Pr₄N)₂CO₃,(n-Bu₄N)₂CO₃, (i-Bu₄N)₂CO₃, or (t-Bu₄N)₂CO₃.

Without wishing to be bound by any particular theory, the use ofbicarbonate, carbonate, and/or ammonium salts may aid in decreasing therate of competing reactions such as hydrolysis during nucleophilicfluorination of an imaging agent precursor.

In some embodiments, the reagent is a salt comprising a cation thatforms a weakly coordinating salt with a fluoride species. As usedherein, a “cation that forms a weakly coordinating salt with a fluoridespecies” refers to a cation that renders a fluoride species reactivewithin a fluorination reaction. For example, the cation may not stronglybind to the fluoride species, allowing the fluoride species to act as anucleophile during a nucleophilic fluorination reaction. Those ofordinary skill the art would be able to select an appropriate cationthat would be suitable as a weakly coordinating counter ion for afluoride species. For example, the cation may be have a relatively largeatomic radius and/or may be a weak Lewis base. In some cases, the cationmay be selected to be lipophilic. In some cases, the cation may compriseone or more alkyl groups. Examples of weakly coordinating cationsinclude cesium ions, ammonium ions, weakly coordinating salts ofhexamethylpiperidindium, S(NMe₂)₃, P(NMe₂)₄, tetraaalkylphosphoniumsalts, tetraarylphosphonium salts, (e.g. tetraphenylphosphonium),hexakis(dimethylamino)diphosphazenium, and tris(dimethylamino)sulfonium.

In some embodiments, the reagent is an ammonium salt, i.e., a saltcomprising a substituted or unsubstituted ammonium ion. In some cases,the ammonium ion is a weakly coordinating cation. In some cases, theammonium salt has the formula, R₄NX, where each R can be the same ordifferent and is alkyl, heteroalkyl, aryl, heteroaryl, or heterocyclic,each optionally substituted, and X is a negatively charged counter ion.In some cases, R is alkyl, heteroalkyl, aryl, heteroaryl, orheterocyclic, each optionally substituted. The ammonium salt may includea range of negatively charged counter ions, including halides,carbonates, and bicarbonates. Examples of ammonium salts include, butare not limited to, ammonium bicarbonate salts, ammonium hydroxidesalts, ammonium acetate salts, ammonium lactate salts, ammoniumtrifluoroacetate salts, ammonium methanesulfonate salts, ammoniump-toluenesulfonate salts, ammonium nitrate salts, ammonium halide salts(e.g., ammonium iodide salts), and ammonium bisulfate salts.

In one set of embodiments, the ammonium salt is a tetraalkylammoniumsalt, such as a tetraalkylammonium bicarbonate salt. For example, theammonium salt may have the formula, R₄NHCO₃, wherein each R isindependently alkyl. In some cases, R is optionally substituted. In someembodiments, the alkyl group is a lower C₁-C₆ alkyl group. In someembodiments, the tetraalkylammonium salt is a basic tetraalkylammoniumsalt.

The salt (e.g., bicarbonate salt and/or ammonium salt) may be utilizedin the reaction such that the molar ratio of the salt to the imagingagent precursor is less than or equal to about 10:1, or less than orequal to about 9:1, or less than or equal to about 8:1, or less than orequal to about 7:1 or less than or equal to about 6:1, or less than orequal to about 5:1, or less than or equal to about 4:1, or less than orequal to about 3:1, or less than or equal to about 2:1, or less than orequal to about 1:1. In some cases, the molar ratio of the salt to theimaging agent precursor is between about 3:1 and about 8:1, or betweenabout 4:1 and about 7:1, or between about 5:1 and about 7:1, or betweenabout 5:1 and about 8:1.

In some embodiments, the reagent is used in combination with a speciescapable of enhancing the reactivity of the fluoride species or otherwisefacilitating conversion of the imaging agent precursor to the imagingagent. For example, the species may be a compound capable of chelatingone or more ions (e.g., metal ions) that may be present within thereaction mixture. Without wishing to be bound by theory, the species maybe used to chelate a counter ion to a fluoride species, such as apotassium ion, thereby increasing the reactivity (e.g., nucleophilicity)of the fluoride species. In certain embodiments, the reagent is used incombination with a multidentate ligand, such as a crown ether or acryptand that is capable of chelating a metal ion. The multidentateligand (e.g., cryptand) may be selected based on the metal ion to bechelated. The multidentate ligand may be, for example,4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (e.g.,Kryptofix® 222). Other cryptands will be known to those of ordinaryskill in the art.

Some embodiments involve the use of a carbonate salt in combination with4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane. In aspecific embodiment, potassium carbonate is used in combination with4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane.

In another set of embodiments, it may be advantageous to utilize themethods described herein in the absence of a cryptand. The term“cryptand” is given its ordinary meaning in the art and refers to a bi-or a polycyclic multidentate ligand for a cation. For example, themethod may be carried out using an ammonium salt, in the absence of acryptand (e.g.,4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane). In somecases, cryptands may increase the pH of the reaction solution, which inthe presence of another reagent (e.g. carbonate salt) may adverselyaffect the yield and/or purity of the fluorination reaction.Accordingly, carrying out the fluorination reaction, in the absence of acryptand, and optionally in the presence of another reagent (e.g.,ammonium and/or bicarbonate salt) may increase the yield and/or purityof the reaction, as described herein.

In another set of embodiments, the method is performed in the absence ofa carbonate salt.

In some embodiments, the use of a salt in the reaction increases theyield by about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 100%, about 200%, about300%, about 400%, about 500%, or greater, relative to conducting thereaction under essentially the same conditions but in the absence of asalt.

As will be understood by one of ordinary skill in the art, duringfluorination, any associated anionic species (e.g., in instances wherethe starting material is a salt) may be exchanged. That is, the startingmaterial may be provided as a first salt (e.g., trifluoroacetate,chloride), and the isolated product (e.g., the fluorinated product) maybe isolated as a second, different salt (e.g., formate, ascorbate,citrate, or trifluoroacetate). In some cases, following formation of asalt, the counter anion may be exchanged in an additional reaction step.For example, the HCl salt of a compound may be exposed to a suitablereagent (e.g., AgOAc or AgOBz) such that the compound forms thecorresponding salt of the reagent (e.g., acetate salt or benzoate salt,respectively). As another example, the TFA salt of a compound may beexposed to a suitable reagent (e.g., phosphoric acid or methanesulfonicacid) such that the compound forms the corresponding salt of the reagent(e.g., phosphate salt or methanesulfonate salt, respectively). Theintermediate salt (e.g., trifluoroacetate salt or chloride salt in theabove-examples) may or may not be isolated prior to exposure to thereagent.

Those of ordinary skill in the art will be able to select and/ordetermine the appropriate set of reaction conditions (e.g.,concentration, temperature, pressure, reaction time, solvents) suitablefor use in a particular application. The imaging agent may be furtherprocessed using one or more purification techniques, and may optionallybe combined with additional components, such as a stabilizing agent.

In some embodiments, the imaging agent is formed as a salt (e.g., apharmaceutically acceptable salt).

In some embodiments, a formate salt comprising formula (VI):

wherein X^(⊖) is formate, is provided.

In other embodiments, an ascorbate salt comprising formula (VII):

wherein X^(⊖) is ascorbate, is provided.

In other embodiments, a citrate salt comprising formula:

wherein X^(⊖) is citrate, is provided.

In other embodiments, a trifluoroacetate salt comprising formula:

wherein X^(⊖) is trifluoroacetate, is provided.

In certain embodiments, the fluorine in the salt of formula (I), (VI),(VII), (IX), or (X) is isotopically enriched with ¹⁸F. In someembodiments, a pharmaceutically acceptable composition is provided.

In certain embodiments, the pharmaceutically acceptable compositioncomprises a salt comprising formula (VI):

wherein X^(⊖) is formate, or a salt comprising formula (VII):

wherein X^(⊖) is ascorbate, or combinations thereof, and optionally apharmaceutically acceptable excipient. Other pharmaceutically acceptablecompositions comprise the citrate salt of formula (IX) or thetrifluoroacetate salt of formula (X).

Pharmaceutically acceptable excipients and other aspects ofpharmaceutically acceptable compositions are described herein.

The formate salt and ascorbate salt have been found to have unexpectedproperties, including improved purity and/or stability as compared toother salt forms of the compound comprising formula (VIII):

wherein X^(⊖) is a counter anion.

In addition, in some cases, it has been found that the salt form of theprecursor of the compound of formula (VI) or (VII) may influence thepurity of the final product in a pharmaceutically acceptable composition(e.g., for use as an imaging agent). For example, with respect to theformate salt (i.e., a compound of formula (VI)), this salt form has beenfound to have unexpected characteristics with respect to purification(e.g., the compound may be isolated in greater ease and/or in higheryields as compared to other salt forms). This may be due to thesolubility characteristics of the salt. In addition, the salt form hasbeen found to have unexpected characteristics with respect to stability.In some embodiments, the ascorbate salts of imaging agents isotopicallyenriched in ¹⁸F are substantially more stable as compared to other saltforms.

In some embodiments, conversion of a compound of formula (VIII) into asuitable compound for use in a pharmaceutically acceptable compositioninvolves three steps: (1) purification (e.g., by HPLC), (2) solventexchange, and (3) formulation. In some cases, the compound of formula(VIII) is purified by HPLC, and the purification, retention, and/orresolution the compound is sensitive to pH and/or buffer capacity of themobile phase. Various reagents may be contained in the mobile phase toeffectively purify the compound, including acetic, citric, and/or formicacid modifiers. In a particular embodiment, the presence of formic acidin the mobile phase is particularly effective. In addition, the additivewas also found to influence solvent exchange, as elution of a compound(e.g., through a C-18 Sep-Pak®) can depend on composition of the mobilephase. In some cases, formulation of the salt can be influenced by boththe pH of the solution and salt form identity. pH can be adjusted tomanage acute radiolytic decomposition during solvent exchange, whilecounter anion selection may be based on long-term antioxidant capacity.

Those of ordinary skill in the art would be able to select a source of afluoride species suitable for use in the methods described herein. Theterm “fluoride species” as used herein refers to a fluoride atom orgroup of atoms comprising at least one fluoride atom, wherein thefluoride atom is capable of reacting with another compound (e.g., animaging agent precursor). In some embodiments, an isotopically-enriched¹⁸F species may be produced by the nuclear reaction ¹⁸O(p,n)¹⁸F fromproton bombardment of [¹⁸O]H₂O in a cyclotron. The method may involvetreating a solution of the ¹⁸F species to remove any impurities, such asunreacted [¹⁸O]H₂O. For example, a solution of the ¹⁸F species may befiltered through an anion exchange column, where the ¹⁸F species isretained on the cationic resin matrix while the [¹⁸O]H₂O is eluted. The¹⁸F species is then removed by washing the anion exchange column withvarious mixtures of solvents and optional reagents (e.g., salt), formingan ¹⁸F-containing solution. In some cases, the anion exchange column iswashed with an aqueous solution of a salt, such as K₂CO₃ or Et₄NHCO₃. Inother cases, the column is washed (e.g., with aqueous K₂CO₃), and theresulting solution diluted (e.g., with MeCN) and/or concentrated (e.g.,to dryness using elevated temperature and/or reduced pressure).Anhydrous [¹⁸F]KF and/or [¹⁸F]Et₄NF may be obtained and reacted with acompound or a salt thereof.

In some cases, the ¹⁸F-containing solution is combined with additionalcomponents prior to reaction with an imaging agent precursor. Forexample, one or more solvents may be added to dilute the ¹⁸F-containingsolution to a desired concentration. In certain embodiments, the¹⁸F-containing solution is diluted with acetonitrile (MeCN). In certainembodiments, the ¹⁸F-containing solution is diluted with acetonitrile(MeCN) and t-BuOH.

In some cases, the ¹⁸F-containing solution may be concentrated todryness by exposure to elevated temperature and/or reduced pressure toform an anhydrous ¹⁸F-containing solid. In some embodiments, the¹⁸F-containing solid may further comprise one or more reagents (e.g.,salts). The chemical composition of the ¹⁸F-containing solid may dependon the number and kind of reagents used in preparation of the¹⁸F-containing solution. For example, a solution of potassium carbonatemay be used to elute the ¹⁸F species from the anion exchange column,thereby resulting in an ¹⁸F-containing solid comprising [¹⁸F]—KF. Inanother example, a solution of tetraethylammonium bicarbonate is used toelute the ¹⁸F species from the anion exchange column, thereby resultingin an ¹⁸F-containing solid comprising [¹⁸F]-Et₄NF.

In some cases, the solution comprising the ¹⁸F species is heated to atemperature ranging from room temperature to about 200° C. For example,a solution comprising the [¹⁸F]-fluoride may be heated to elevatedtemperatures to encourage evaporation of the solvent (e.g., to about110° C.). In some embodiments, the solution is heated to a temperatureranging from about 90-120° C. or from about 100-150° C. In some cases,the solution is heated to about 75° C., about 85° C., about 95° C.,about 105° C., about 115° C., about 125° C., or greater. In some cases,the solution is placed under a reduced pressure of about 100 mm Hg,about 125 mm Hg, about 150 mm Hg, about 175 mm Hg, about 200 mm Hg,about 225 mm Hg, about 250 mm Hg, about 275 mm Hg, about 300 mm Hg,about 325 mm Hg, about 350 mm Hg, about 375 mm Hg, about 400 mm Hg, orgreater. In some cases, the solution is placed under a reduced pressureof about 100 mbar, about 125 mbar, about 150 mbar, about 175 mbar, about200 mbar, about 225 mbar, about 250 mbar, about 275 mbar, about 280mbar, about 300 mbar, about 325 mbar, about 350 mbar, about 375 mbar,about 400 mbar, about 450 mbar, about 500 mbar, or greater. Those ofordinary skill in the art would be able to select and/or determineconditions suitable for a particular process. In some embodiments, thesolution is concentrated to dryness at about 150 mm Hg and about 115° C.In some embodiments, the solution is concentrated to dryness at about375 mm Hg and about 115° C. In some embodiments, the solution isconcentrated to dryness at about 400 mbar and about 110-150° C. In someembodiments, the solution is concentrated to dryness at about 280 mbarand about 95-115° C.

The fluoride species and/or the reagent, if present, is then contactedwith the imaging agent precursor under conditions that result inconversion of the imaging agent precursor to the imaging agent productvia nucleophilic fluorination. Those of ordinary skill in the art wouldbe able to select conditions suitable for use in a particular reaction.For example, the ratio of fluoride species to imaging agent precursormay be selected to be about 1:10,000 or more, about 1:5000 or more,about 1:3000 or more, about 1:2000 or more, about 1:1000 or more, about1:500 or more, about 1:100 or more, about 1:50 or more, about 1:10 ormore, about 1:5 or more, or, in some cases, about 1:1 or more. In someembodiments, the fluoride species may be present at about 10 mol %, orabout 5 mol %, or about 3 mol %, or about 2 mol %, or about 1 mol % orabout 0.5 mol %, or about 0.1 mol %, or about 0.05 mol %, or about 0.01mol % relative to the amount of imaging agent precursor. In someembodiments, the fluoride species is isotopically enriched with ¹⁸F. Forexample, the ratio of ¹⁸F species to imaging agent precursor may beselected to be about 1:1,000,000 or more, or about 1:500,000 or more, orabout 1:250,000 or more, or about 1:100,000 or more, or about 1:50,000or more, or about 1:25,000 or more, or about 1:10,000 or more, about1:5000 or more, about 1:3000 or more, about 1:2000 or more, about 1:1000or more, about 1:500 or more, about 1:100 or more, about 1:50 or more,about 1:10 or more, about 1:5 or more, or, in some cases, about 1:1 ormore.

In some embodiments, the nucleophilic fluorination reaction is carriedout in the presence of one or more solvents, for example, an organicsolvent, a non-organic solvent (e.g., an aqueous solvent), or acombination thereof. In some cases, the solvent is a polar solvent or anon-polar solvent. In some embodiments, the solvent is an aqueoussolution, such as water. The solvent comprises at least about 0.001%water, at least about 0.01% water, at least about 0.1% water, at leastabout 1% water, at least about 5%, at least about 10%, at least about20% water, at least about 30% water, at least about 40% water, at leastabout 50% water, or greater. In some cases, the solvent may comprisebetween about 0.1% and about 100% water, about 1% to about 90%, about 1%to about 70%, about 1% to about 50%, or about 10% to about 50%. In somecases, the solvent comprises no more than about 10% water, about 5%water, about 4% water, about 3% water, about 2% water, about 1% water,or about 0.5% water. In some cases, the solvent comprises between about0.01% water and about 5% water, or between about 0.01% water and about2% water, or between about 0.1% water and about 0.2% water.

Other non-limiting examples of solvents useful in the methods include,but are not limited to, non-halogenated hydrocarbon solvents (e.g.,pentane, hexane, heptane, cyclohexane), halogenated hydrocarbon solvents(e.g., dichloromethane, chloroform, fluorobenzene,trifluoromethylbenzene), aromatic hydrocarbon solvents (e.g., toluene,benzene, xylene), ester solvents (e.g., ethyl acetate), ether solvents(e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane), andalcohol solvents (e.g., ethanol, methanol, propanol, isopropanol,tert-butanol). Other non-limiting examples of solvents include acetone,acetic acid, formic acid, dimethyl sulfoxide, dimethyl formamide,acetonitrile, p-cresol, glycol, petroleum ether, carbon tetrachloride,hexamethyl-phosphoric triamide, triethylamine, picoline, and pyridine.In some embodiments, the reaction is carried out in a polar solvent,such as acetonitrile. In some cases, the solvent may be selected so asto reduce and/or minimize the formation of side products. In certainembodiments, the fluorination reaction is carried out in MeCN as thesolvent. In certain embodiments, the fluorination reaction is carriedout in t-BuOH as the solvent. In certain embodiments, the fluorinationreaction is carried out in a mixture of MeCN and t-BuOH as the solvent.In certain embodiments, the fluorination reaction is carried out in DMFas the solvent. In certain embodiments, the fluorination reaction iscarried out in DMSO as the solvent. In certain embodiments, thefluorination reaction is carried out in THF as the solvent.

In certain embodiments, an anhydrous ¹⁸F-containing solid, optionallycomprising a reagent, may be contacted with a solution of an imagingagent precursor (e.g., a tosylate precursor), and the resulting solutionis heated to an elevated temperature for a select period of time. Thesolution may be, for example, an acetonitrile solution. In otherembodiments, a solution of the ¹⁸F species and reagent, if present, iscontacted with a solid imaging agent precursor or a solution of theimaging agent precursor.

Some embodiments involve contacting the imaging agent precursor with thefluoride species in a solution having a pH below about 13, below about12, or below about 11. In some cases, the solution has a pH betweenabout 8 and about 9, or between about 8 and about 10, or between about 7and about 8. In certain embodiments, the pH range for the fluorinationreaction is greater than about 6, or greater than about 7, or betweenand including 7-13, between and including 6-12, between and including7-12, between and including 8-12, between and including 9-12, andbetween and including 10-12.

In some cases, the solution comprising the ¹⁸F species, imaging agentprecursor, and, optionally, reagent, is heated to an elevatedtemperature for a period of time. For example, the solution may beheated to about 50° C., about 60° C., about 70° C., about 80° C., about90° C., about 100° C., about 110° C., about 120° C., about 150° C.,about 170° C., about 200° C., about 225° C., about 250° C., or greater,for a period of about 5 minutes or less, about 10 minutes or less, about20 minutes or less, about 30 minutes or less. It should be understoodthat other temperatures and reaction times may be used. Upon completionof the reaction, the reaction mixture is cooled (e.g., to roomtemperature) and optionally diluted with a solvent, such as water, ormixtures of solvents, such as water/acetonitrile. In some embodiments,the reaction mixture is heated to elevated temperatures to encourageevaporation of the solvent (e.g., to about 95° C.). In some embodiments,the solution is heated to a temperature ranging from about 55-125° C. Insome cases, the solution is heated to about 65° C., about 75° C., about85° C., about 95° C., about 105° C., about 115° C., or greater. In somecases, the solution is placed under a reduced pressure of about 100 mmHg, about 125 mm Hg, about 150 mm Hg, about 175 mm Hg, about 200 mm Hg,about 225 mm Hg, about 250 mm Hg, about 275 mm Hg, about 300 mm Hg,about 325 mm Hg, about 350 mm Hg, about 375 mm Hg, about 400 mm Hg, orgreater. In some cases, the solution is placed under a reduced pressureof about 100 mbar, about 125 mbar, about 150 mbar, about 175 mbar, about200 mbar, about 225 mbar, about 250 mbar, about 275 mbar, about 280mbar, about 300 mbar, about 325 mbar, about 350 mbar, about 375 mbar,about 400 mbar, about 450 mbar, about 500 mbar, or greater. Those ofordinary skill in the art would be able to select and/or determineconditions suitable for a particular process. In some embodiments, thesolution is concentrated to dryness under a flow of inert gas at about95° C.

Upon completion of the fluorination reaction, the resulting imagingagent is optionally subjected to one or more purification steps. In somecases, the imaging agent may be reconstituted in a solvent prior topurification (e.g., by chromatography such as HPLC). In some cases, theimaging agent is dissolved in water, acetonitrile, or combinationsthereof. In some embodiments, following formation of a solutioncomprising the imaging agent and the solvent and prior to purification(e.g., by HPLC), the solution is heated. In a particular embodiment, theimaging agent is reconstituted in a water/acetonitrile mixture andheated (e.g., to a temperature of about 90-100° C.) for about 1 minute,about 3 minutes, about 5 minutes, about 10 minutes, about 20 minutes,about 30 minutes, or more. Following the heating of the mixture, thesolution may be optionally cooled prior to purification.

Deprotection

Those of ordinary skill in the art will be aware of suitable conditionsfor deprotecting guanidine functional groups. As discuss below, theprotecting groups may be removed before or after fluorination. In someembodiments, the suitable conditions comprise exposing a compoundcomprising a protected guanidine functional group to an acid. The acidmay be added neat or in a solution (e.g., such that the acid is at aconcentration of about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M,about 0.5 M, about 0.75 M, or about 1.0 M). In certain embodiments, thenitrogen-protecting group is t-butyloxycarbonyl, and the acid used forthe deprotecting step is trifluoroacetic acid. In certain embodiments,following deprotection, the compound is a salt (e.g., a trifluoroacetatesalt).

In some cases, the suitable conditions for deprotection comprise acidicconditions. The acid may be provided at a ratio of about 2:1, about 1:1,about 1:2, about 1:3, or about 1:4 compound:acid. In certainembodiments, the pH range for deprotection of imaging agent precursorssuch as compounds of Formula (II) (or alternatively of protectedfluorinated imaging agents of the invention) may be equal to or lessthan about 4, including equal to or less than about 3, equal to or lessthan about 2, and equal to or less than about 1.

The conditions may comprise one or more solvents. Non-limiting examplesof solvents are provided herein. The reaction may be carried out at anysuitable temperature, and in certain embodiments, the deprotectionreaction is carried out at room temperature or above room temperature.The product may be analyzed, isolated, and/or purified using techniquesknown to those of ordinary skill in the art (e.g., columnchromatography, HPLC, NMR, MS, IR, UV/Vis). In some cases, the productis isolated as a salt (e.g., via filtration, crystallization). Incertain embodiments, the salt is an ascorbate salt. In certainembodiments, the salt is a formate salt. In other embodiments, the saltis a citrate salt or a trifluoroacetate salt.

Purification and Formulation

In some cases, the synthesis, purification, and/or formulation of animaging agent (e.g., a compound comprising formula (I) or (V)) isperformed using an automated reaction system optionally comprising acassette, wherein the cassette comprises a synthesis module, and/or apurification module, and/or a formulation module. Automated reactionsystems and cassettes are described herein.

Purification and isolation may be performed using methods known to thoseskilled in the art, including separation techniques like chromatography,or combinations of various separation techniques known in the art, forexample, extractions, distillation, and crystallization. In oneembodiment, high performance liquid chromatography (HPLC) is used with asolvent, or mixture of solvents, as the eluent, to recover the product.In some cases, the eluent includes a mixture of water and acetonitrile,such as a 20:80 water:acetonitrile mixture. The content of water in theeluent may vary from, for example, about 1% to about 30%. In some cases,HPLC purification may be performed using a C18 column. The product maybe analyzed (e.g., by HPLC) to determine yield (e.g., radiochemicalyield) and/or radiochemical purity. The radiochemical purity may begreater than about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 97%, about 98%, about 99%, or more. The percent yieldof a product may be greater than 10%, greater than 20%, greater than30%, greater than 40%, greater than 50%, greater than about 60%, greaterthan about 70%, greater than about 75%, greater than about 80%, greaterthan about 85%, greater than about 90%, greater than about 92%, greaterthan about 95%, greater than about 96%, greater than about 97%, greaterthan about 98%, greater than about 99%, or greater. In some embodiments,the radiochemical yield ranges from 15-50%.

The product may be further processed using additional purificationtechniques, such as filtration. In some cases, the imaging agent ispurified using HPLC, to produce a solution of HPLC mobile phase and theimaging agent. The HPLC mobile phase may be subsequently exchanged for asolution of ascorbic acid or a salt thereof, and ethanol solution, byfiltration through a C-18 resin (e.g., C18 Sep-Pak® cartridge). In someembodiments, the solution of the HPLC mobile phase and the imaging agentis filtered through a C-18 resin, where the imaging agent remains on theresin and the other components, such as acetonitrile and/or othersolvents or components, are removed via elution. The C-18 resin may befurther washed with a solution of ascorbic acid or a salt thereof, andthe filtrate discarded. To recover the purified imaging agent, the C-18resin is washed with a solvent, such as ethanol, and the resultingsolution is optionally further diluted with an ascorbic acid solution ora salt thereof, as described herein.

Optionally, the recovered product is combined with one or morestabilizing agents, such as ascorbic acid or a salt thereof. Forexample, a solution comprising the purified imaging agent may be furtherdiluted with a solution of ascorbic acid or a salt thereof. As describedherein, a formulation may be prepared via an automated reaction systemcomprising a cassette.

In some cases, a solution comprising the imaging agent product may besterile filtered (e.g., using a 13 mm diameter, Millipore, Millex PVDF0.22 μm sterilizing filter) into a sterile product vial. The sterileproduct vial may be a commercially available, pre-sterilized unit thatis not opened during the production process, as any imaging agents (orother components) may be aseptically inserted through the septum priorto use. Those of ordinary skill in the art would be able to selectsuitable vials and production components, including commerciallyavailable, pre-sterilized units comprising a 0.22 μm pore size membraneventing filter and quality control sampling syringes.

Following aseptic filtration, individual doses may be filled insyringes, labeled, and shipped to a clinical site. Dosing administrationtechniques, kits, cassettes, methods and systems (e.g., automatedreaction systems) for synthesis of the imaging agent, and testingprocedures are described herein. In some embodiments, the product isdispensed into a 3 or 5 mL syringe and labeled for distribution. Labelsmay be prepared at a radiopharmacy and applied to a syringe shield andshipping container. Additional labels may be provided in the shippingcontainer for inclusion in clinical site records.

Uses of Imaging Agents

In another aspect, the present invention provides methods of imaging,including methods of imaging a subject that includes administering acomposition or formulation that includes an imaging agent of theinvention (i.e., a compound of Formula (I), including a compound ofFormula (V), such as, but not limited to, imaging agent-1) to thesubject by injection, infusion, or any other method of administration,and imaging a region of interest of the subject. Regions of interest mayinclude, but are not limited to, the heart, a portion of the heart, thecardiovascular system, cardiac vessels, blood vessels (e.g., arteriesand/or veins), brain, pancreas, adrenal glands, other organs, andtumors. As described herein, imaging agent-1 comprises the formula:

or a pharmaceutically acceptable salt, free base, or combinationthereof. In some embodiments, a pharmaceutically acceptable salt ofimaging agent-1 comprises the formula:

wherein X^(⊖) is a counter anion. In certain embodiments, X^(⊖) isformate or ascorbate. In some embodiments, the X^(⊖) is citrate ortrifluoroacetate.

In some embodiments, methods of this disclosure include (a)administering to a subject a composition that includes an imaging agentof the invention including, but not limited to, imaging agent-1, and (b)acquiring at least one image of at least a portion of the subject. Insome cases, the step of acquiring employs positron emission tomography(PET) for visualizing the distribution of the imaging agent within atleast a portion of the subject. As will be understood by those ofordinary skill in the art, imaging using methods of this disclosure mayinclude full body imaging of a subject, or imaging of a specific bodyregion, organ, or tissue of the subject that is of interest. Forexample, if a subject is known to have, or is suspected of havingmyocardial ischemia, methods of this disclosure may be used to image theheart of the subject. In some embodiments, imaging may be limited to theheart or may include the heart and its associated vasculature.

In some embodiments, imaging agents of the invention, including but notlimited to imaging agent-1, are used to monitor and/or assess certainaspects of the sympathetic nervous system (SNS). The SNS plays a role innormal cardiac regulation and/or the pathogenesis of heart failuredevelopment and/or progression. Generally, following myocardial insult(e.g., myocardial infarction, valve regurgitation, hypertension),compensatory activation of the SNS is induced to help maintainsufficient cardiac output. Sustained elevation of the cardiac SNS cancause elevated cardiac norepinephrine (NE) release, down regulation ofthe beta1 adrenergic receptor, and/or down regulation of the NEtransporter (NET), which can result in spillover of NE. Elevated levelsof NE can be attributed to cardiac myocyte hypertrophy, fibroblastactivation, collagen deposition, and/or myocyte apoptosis, which canresult in ventricle remodeling and/or susceptibility to arrhythmia.

In some embodiments, assessment of the changes and/or the presence of aneurotransmitter in a subject, and certain parameters relating to theneurotransmitter provides feedback relating to cardiac events. Forexample, assessment of NET in a subject can be used to provide feedbackrelating to cardiac events and/or cardiac exposure to NE. In some cases,the neurotransmitter is a monoamine other than NE.

In some embodiments, the neurotransmitter is NE. Utilizing an imagingagent that targets NET permits imaging of the location, concentration,density, and/or distribution of NETs and also can be used to detectchanges in NETs over time, for example, by acquiring a first NET imagein a subject or region of a subject; obtaining a subsequent NET image ofthe subject or the region of the subject and comparing the first andsubsequent images. Differences between the images can provideinformation on the change in NET status in the subject or region of thesubject. Changes in a NET parameter (e.g., location, density,concentration, and/or distribution) over time may be assessed andcorrelated with disease onset, progression, and/or regression. In someembodiments, a method comprises administering a dose of apharmaceutically acceptable composition (e.g., imaging agent-1) to asubject, and acquiring at least one image of a portion of the subject,wherein the image allows for the assessment and/or detection of NET inthe subject. In some cases, the detection comprises detection of thelevel (e.g., concentration) of NET, detection of the density of NET,detection of NET function, and/or detection of the localization of NET.

In some embodiments, changes in NET (e.g., density, localization,concentration, function) may be used to assess the presence and/orabsence of a condition, disease, and/or disorder. For example, in somecases, changes in NET may be used to assess cardiac sympatheticinnervation and/or myocardial sympathetic function in a subject. Forexample, an increase or decrease in NET concentration in a portion ofthe subject (e.g., heart) may indicate the cardiac sympatheticinnervation in that portion of the subject. In some cases, subjects withimpaired NET functions are correlated with heart failure and/or rapidmyocardial reorganization.

In some embodiments, an imaging agent that targets NET may also be usedto observe, estimate and/or quantify localized blood flow to tissue.More specifically, there may be instances in which the level of imagingagent (or radioactivity) observed in the myocardium, is decreasedcompared to normal or below threshold. There may be various causes ofthis decreased signal, one of which may be reduced blood flow to andthrough the myocardium. In order to determine the cause, the subject maybe imaged using a different imaging agent and/or a different imagingmodality suitable for detecting blood flow. Comparison of imagesobtained using the different methods can reveal whether the decrease orabsence of signal from the imaging agent that targets NET isattributable to blood flow rather than to a difference in NET level,activity and the like. In other embodiments of the invention, themyocardium may be imaged serially, for example immediately afteradministration of the imaging agent, in order to observe movement of theimaging agent into the heart. Such serial images should yieldinformation about blood flow through the heart. Later images are alsoobtained as these reveal a more steady state of blood flow into and outof the heart as well as blood retention in the heart. In this way,alterations in global, local, or regional blood flow may bedistinguished from local or regional changes in NET density,localization, concentration, and function as described above. In someembodiments, an imaging agent that targets NET is used to assess theability of a therapeutic agent and/or treatment to modify NET. Forexample, images acquired from a subject administered an imaging agent ofthe including but not limited to imaging agent-1 before therapeutictreatment can be compared to images acquired from the same subject aftertherapeutic treatment of the subject to determine if the treatment hasaffected the location, concentration, and/or density of NET for thesubject. Similarly, images at different times and/or before and aftertreatment can be used to detect changes in NET in a subject over timeand/or with treatment.

In some aspects, global images (e.g., global NET images) are acquired,and in other aspects of the invention, regional images (e.g., regionalNET images) are acquired following administration of an imaging agentthat targets NET, wherein a global image is an image of all orsubstantially all of an organ (e.g., heart, kidney, pancreas), and aregional image is an image of only a portion of an organ. Images can beacquired using an image collection system such as a PET system, a SPECTsystem, or any other suitable imaging system.

In some embodiments, images may be acquired over a single time interval,and in other embodiments, they may be acquired as a series of images ofthe same or different acquisition durations beginning either at the timeof administration or at a later time.

In some embodiments, methods of diagnosing or assisting in diagnosing adisease or condition, assessing efficacy of a treatment of a disease orcondition, or imaging of a subject with a known or suspectedcardiovascular disease or condition changing sympathetic innervationsare provided. A cardiovascular disease can be any disease of the heartor other organ or tissue supplied by the vascular system. The vascularsystem includes coronary arteries, and all peripheral arteries supplyingthe peripheral vascular system and the brain, as well as veins,arterioles, venules, and capillaries. In cases, cardiac innervation maybe examined, as abnormalities in cardiac innervation have beenimplicated in the pathophysiology of many heart diseases, includingsudden cardiac death, congestive heart failure, diabetic autonomicneuropathy, myocardial ischemia, and cardiac arrhythmias. Othernon-limiting examples of cardiovascular diseases of the heart includediseases such as coronary artery disease, myocardial infarction,myocardial ischemia, angina pectoris, congestive heart failure,cardiomyopathy (congenital or acquired), arrhythmia, or valvular heartdisease. In some embodiments, the methods disclosed herein are usefulfor monitoring and measuring cardiac innervation. For example, a methoddescribed herein can determine the presence or absence of cardiacinnervation. Conditions of the heart may include damage, not brought onby disease but resulting from injury e.g., traumatic injury, surgicalinjury. Methods described herein can be used in some embodiments todetermine global or regional changes in cardiac sympathetic innervation.

In some cases, a subject whom an imaging agent of the invention may beadministered may have signs or symptoms suggestive of a disease orcondition associated with abnormalities in cardiac innervation. In somecases, use of the imaging agent can be used to diagnose early orpre-disease conditions that indicate that a subject is at increased riskof a disease. Imaging methods described herein may be used to detectcardiac innervation in subjects already diagnosed as having a disease orcondition associated with abnormalities in cardiac innervation, or insubjects that have no history or diagnosis of such a disease orcondition. In other instances, the methods may be used to obtainmeasurements that provide a diagnosis or aid in providing a diagnosis ofa disease or condition associated with abnormalities in cardiacinnervation. In some instances, a subject may be already undergoing drugtherapy for a disease or condition associated with abnormalities incardiac innervation, while in other instances a subject may be withoutpresent therapy for a disease or condition associated with abnormalitiesin cardiac innervation. In some embodiments, the method may be used toassess efficacy of a treatment for a disease or condition. For example,the heart can be visualized using contrast/imaging agents describedherein before, during, and/or after treatment of a condition affectingthe heart of a subject. Such visualization may be used to assess adisease or condition, and aid in selection of a treatment regimen, e.g.therapy, surgery, medications, for the subject.

In some embodiments, a compound of the present invention is employed fordetermining the presence or absence of a tumor in a subject. In someembodiments, the tumor is a NET-expressing tumor. In some embodiments,an imaging agent of the invention is employed for determining theresponse to therapy of a tumor in a subject. Methods for determining thepresence of a tumor and/or for determining the response to therapy of atumor in a subject can follow the same or similar methods as describedfor methods of imaging a subject.

In some embodiments, an imaging agent of the invention (e.g., imagingagent-1) is used as an imaging agent in combination with positronemission tomography (PET) or with other imaging methods including, butnot limited to, single photon emission computed tomography (SPECT)imaging. In some cases, PET imaging may be used in cardiac sympatheticneuronal imaging in a subject following administration of imagingagent-1 to the subject. For example, imaging agent-1 may be administeredto a subject and imaged in the subject using PET. As will be known tothose of ordinary skill in the art, PET is a non-invasive technique thatallows serial images and measurements to be obtained in a single subjectover a time period. PET imaging used may be carried out using knownsystems, methods, and/or devices. In some embodiments, PET imaging isconducted using a cardiac imaging system. A cardiac imaging system mayinclude PET imaging functionality; and a control unit configured todrive the imaging functionality to perform a PET imaging procedure on aportion of the subject of interest before, during and/or afteradministration of imaging agent-1 to the subject. In some cases, thecontrol unit is configured to drive the imaging functionality to performa PET imaging procedure. The control unit may comprise a computer systemand/or software. In such a case, the computer system may be programmedor configured to execute the required methods for acquiring and/oranalyzing the images. Further, the system may include a data storagedevice that is readable by a machine, embodying a set of instructionsexecutable by the machine to perform the required methods of acquiringand/or analyzing the images.

Imaging systems (e.g., cardiac imaging systems) and components thereofwill be known to those of ordinary skill in the art. Many imagingsystems and components (e.g., cameras, software for analyzing theimages) are known and commercially available, for example, a SiemensBiograph-64 scanner or other scanner suitable for imaging. In someembodiments, image data is acquired in list mode, and such list data maybe used to create static, dynamic, or gated images. An appropriateperiod of time for acquiring images can be determined by one of ordinaryskill in the art, and may vary depending on the cardiac imaging system,the imaging agent (e.g., amount administered, composition of the imagingagent, subject parameters, area of interest). As used herein a “periodof acquiring images” or an“image acquisition period” may be a period oftime for obtaining a single continuous image, and/or may be a periodduring which one or more individual discrete images are obtained. Thus,a period of image acquisition can be a period during which one or moreimages of one or more regions of a subject are acquired.

The term “list mode,” as used herein, is given its ordinary meaning inthe art. With respect to PET, list mode is a form in which the data thatis used to create a PET image can be initially collected. In list mode,each of or a portion of coincidence events (i.e., each of a portion ofdetected photon pairs) generates an entry in a list of events. Eachentry includes various information including, but not limited to, whichdetectors were involved, the energy of the photons detected, the time ofdetection, and/or whether there was a cardiac gating mark. Theinformation can be converted into one or more images by the process ofrebinning and/or histogramming, in which all or a portion of the eventsfor each pair of detectors is summed, followed by the resulting set ofprojections (e.g., in the form of a sinogram wherein for each slice,each horizontal line in the sinogram represents the projections forcoincidences at a given angle). List mode may be contrasted with“histogram mode” in which the summations are completed duringacquisition so that the only raw data is the sinogram. In someembodiments, histogram mode may be employed.

In some embodiments, a period of image acquisition after administrationof imaging agent-1 to a subject may be between about 0 seconds and about60 minutes, between about 1 minute and about 30 minutes, between about 5minutes and about 20 minutes, or at least about 1 minute, at least about3 minutes, at least about 5 minutes, at least about 6 minutes, at leastabout 7 minutes, at least about 8 minutes, at least about 9 minutes, atleast about 10 minutes, at least about 15 minutes, at least about 20minutes, at least about 30 minutes, at least about 45 minutes, at leastabout 60 minutes, at least about 90 minutes, at least about 2 hours, atleast about 3 hours, at least about 4 hours, at least about 5 hours, orgreater. In some embodiments, a period of image acquisition may beginprior to administration of imaging agent-1 to a subject. For example, aperiod of image acquisition may begin more than about 10 minutes, about5 minutes, about 4, minutes, about 3 minutes, about 2 minutes, about 1minute, about 0 minutes prior to administration of imaging agent-1 tothe subject. In some embodiments, imaging may be continuous over theimaging period of time, or images may be acquired at intervals such asin periodic or gated imaging.

In some embodiments, an imaging agent of the invention (e.g., imagingagent-1) is provided in ethanol/ascorbic acid. In some embodiments, animaging agent of the invention (e.g., imaging agent-1) is provided as acomposition comprising ethanol, ascorbic acid (e.g., as sodiumascorbate), and water. In some cases, the composition comprises lessthan about 20 weight % ethanol, less than about 15 weight % ethanol,less than about 10 weight % ethanol, less than about 8 weight % ethanol,less than about 6 weight % ethanol, less than about 5 weight % ethanol,less than about 4 weight % ethanol, less than about 3 weight % ethanol,or less ethanol. In some cases, the composition comprises less thanabout 100 mg/mL, less than about 75 mg/mL, less than about 60 mg/mL,less than about 50 mg/mL, less than about 40 mg/mL, less than about 30mg/mL, less than about 20 mg/mL, less than about 10 mg/mL, or lessascorbic acid (e.g., sodium ascorbate) in water. A non-limiting,exemplary formulation of imaging agent-1 includes about 5 weight %ethanol and about 50 mg/ml ascorbic acid. In a particular non-limitingembodiment, a compound comprising formula (VI) or (VII) is provided as asolution in water comprising less than about 5 weight % ethanol and lessthan about 50 mg/mL sodium ascorbate in water. As will be understood bythose of ordinary skill in the art, in the presence of ascorbic acid, atleast a portion of the imaging agent-1 may be present as the ascorbatesalt such that imaging agent-1 has the formula:

wherein X^(⊖) is ascorbate.

Additional components of a composition comprising an imaging agent ofthe invention (e.g., imaging agent-1) may be selected depending on themode of administration to the subject. Various modes of administrationwill be known to one of ordinary skill in the art which effectivelydeliver the pharmacological agents of the invention to a desired tissue,cell, organ, or bodily fluid. In some embodiments, an imaging agent ofthe invention (e.g., imaging agent-1) is administered intravenously(e.g., intravenous bolus injection) using methods known to those ofordinary skill in the art. As used herein, a dose that is “administeredto a subject” means an amount of the imaging agent, e.g. imagingagent-1, that enters the body of the subject.

In some embodiments, the volume of the administered imaging agent may bebetween 0 and about 3 mL, between about 3 mL and about 5 mL, or betweenabout 5 mL and about 10 mL.

In some embodiments, due to factors such as partial retention of imagingagent such as imaging agent-1 in a syringe, tubing, needles, or otherequipment used to administer the imaging agent to a subject, the amountof an imaging agent such as imaging agent-1 that is measured ordetermined to be in the a syringe or other equipment prepared foradministration may be more than the amount in the dose that isadministered to the subject. In some embodiments, an injection of animaging agent is followed by a flushing injection of normal saline intothe subject, using the same tubing, needle, port, etc., used foradministration of the imaging agent.

Flushing may be performed immediately following administration of theimaging agent-1, or up to about 1 min, about 2 min, about 3 min, about 5min, or more after the administration. In some embodiments, flushing maybe performed between 0 and 10 seconds, between 10 seconds and 25seconds, or between 25 seconds and 60 seconds.

The volume of saline or other agent for flushing may be up to about 5ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about15 ml, about 20 ml, or more. As will be understood by those of ordinaryskill in the art, in embodiments where imaging agent-1 is administeredusing a syringe or other container, the true amount of imaging agent-1administered to the subject may be corrected for any imaging agent-1that remains in the container. For example, the amount of radioactivityremaining in the container, and tubing and needle or delivery instrumentthat carried the imaging agent from the container and into the subjectcan be determined after the imaging agent has been administered to thesubject and the difference between the starting amount of radioactivityand the amount remaining after administration indicates the amount thatwas delivered into the subject. In some cases, the container orinjection device (e.g., catheter, syringe) may be rinsed with a solution(e.g., saline solution) following administration of imaging agent-1.

A composition of an imaging agent of the invention (e.g., imagingagent-1) for injection may be prepared in an injection syringe. Imagingagents may be prepared by a radiopharmacy (e.g., using the methodsdescribed herein) and/or a PET manufacturing center and provided to ahealth-care professional for administration. A dose of imaging agent-1may be diluted with saline (e.g., as described herein), if needed toobtain a practical dose volume. For example, if the activityconcentration of imaging agent-1 is so high that only about 0.1 mL isneeded for an appropriate dose for a subject, the solution can bediluted, e.g., with sterile saline, so the syringe contains about 0.5 mlto about 6 ml or more ml of an imaging agent-1 solution foradministration. In some embodiments, an injection volume for imagingagent-1 is between about 0.5 and about 5 ml, about 1 and about 4 ml,about 2 and about 3 ml, at least about 0.5 ml, about 1 ml, about 2 ml,about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml,about 9 ml, about 10 ml, or more. Those of skill in the art willrecognize how to dilute imaging agent-1 to produce a sufficient dosevolume for administration. In some aspects, imaging agent-1 is providedin a container such as a vial, bottle, or syringe, and may betransferred, as necessary, into a suitable container, such as a syringefor administration.

Components of a composition comprising an imaging agent of the invention(e.g., imaging agent-1) may be selected depending on the mode ofadministration to the subject. Various modes of administration thateffectively deliver imaging agents of the invention to a desired tissue,cell, organ, or bodily fluid will be known to one of ordinary skill inthe art. In some embodiments, the imaging agent is administeredintravenously (e.g., intravenous bolus injection) using methods known tothose of ordinary skill in the art.

The useful dosage of the imaging agent to be administered and theparticular mode of administration will vary depending upon such factorsas age, weight, and particular region to be imaged, as well as theparticular imaging agent used, the diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, microsphere,liposome, or the like, as described herein, and as will be readilyapparent to those skilled in the art.

In one embodiment, imaging agent-1 is administered by intravenousinjection, usually in saline solution, at a dose of between about 0.1and about 20 mCi (and all combinations and subcombinations of dosageranges and specific dosages therein, and as described below), or betweena dose of about 0.5 and about 14 mCi. Imaging is performed usingtechniques well known to the ordinarily skilled artisan and/or asdescribed herein.

Based on dosing studies, the desirable maximum dose administered to asubject may be based on determining the amount of imaging agent of theinvention (e.g., imaging agent-1), which limits the radiation dose toabout 5 rem to the critical organ (e.g., urinary bladder) and/or about 1rem effective dose (ED) or lower, as will be understood by those ofordinary skill in the art. In some embodiments of the invention, themaximum desirable dose or total amount of imaging agent-1 administeredis between about 8 mCi and about 13 mCi. In some embodiments of theinvention, the maximum desirable dose or total amount of imaging agent-1administered is between about 10 mCi and about 13 mCi. In someembodiments of the invention, the maximum desirable dose or total amountof imaging agent-1 administered is between about 8 mCi and about 10 mCi.In some embodiments, a desirable dose may be less than or equal to about15 mCi, less than or equal to about 14 mCi, less than or equal to about13 mCi, less than or equal to about 12 mCi, less than or equal to about11 mCi, or less than or equal to about 10 mCi over a period of time ofup to about 10 minutes, about 30 minutes, about 1 hour, about 2 hours,about 6 hours, about 12 hours, about 24 hours, or about 48 hours. Insome embodiments, the maximum dose of imaging agent-1 administered to asubject may be less than about 14 μg per about 50 kg of body weight perday. That is in some embodiments of the invention, the maximum dose of acomposition comprising imaging agent-1 administered to a subject may beless than about 0.28 μg of a imaging agent-1 per kg of body weight perday.

In some embodiments, the total amount of imaging agent-1 administered toa subject is between about 0.1 mCi and about 30 mCi, or between about0.5 mCi and about 20 mCi. In some embodiments, the total amount ofimaging agent-1 administered to a subject is less than or equal to about50 mCi, less than or equal to about 40 mCi, less than or equal to about30 mCi, less than or equal to about 20 mCi, less than or equal to about18 mCi, less than or equal to about 16 mCi, less than or equal to about15 mCi, less than or equal to about 14 mCi, less than or equal to about13 mCi, less than or equal to about 12 mCi, less than or equal to about10 mCi, less than or equal to about 8 mCi, less than or equal to about 6mCi, less than or equal to about 4 mCi, less than or equal to about 2mCi, less than or equal to about 1 mCi, or less than or equal to about0.5 mCi. The total amount administered may be determine based on asingle dose or multiple doses administered to a subject within a timeperiod of up to or at least about 30 seconds, about 1 minute, about 10minutes, about 30 minutes, about 1 hour, about 2 hours, about 6 hours,about 12 hours, about 24 hours, about 48 hours, or about 1 week.

In some aspects of the invention, between about 10 and about 13 mCi, orbetween about 8 to about 10 mCi of imaging agent-1 is administered to asubject, and a first period of image acquisition begins at the time ofadministration (e.g. injection) or begins at more than about 0 minutes,about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about5 minutes, prior to the administration of the imaging agent-1. In someembodiments of the invention, the first imaging continues for at leastabout 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes,about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes,about 105 minutes, about 120 minutes, or longer. Following the firstperiod of imaging, the subject may undergo one or more additionalimaging acquisition periods during up to about 1, about 2, about 3,about 4, about 5, about 6, or more hours after the administration ofimaging agent-1. One or more additional image acquisition periods mayhave a duration of between about 3 and about 40 minutes, about 5 andabout 30 minutes, about 7 and about 20 minutes, about 9 and about 15minutes, and may be for about 10 minutes. The subject, in someembodiments, may return once, twice, or three or more times foradditional imaging following the first injection of imaging agent-1wherein a second, third, or more, injections of imaging agent-1 may beadministered. A non-limiting example of an administration and imageacquisition method for imaging agent-1 for a subject comprises injectionof between about 10 and about 13 mCi, or between about 8 to about 10 mCiof imaging agent-1 to the subject, with image acquisition starting lessthan about 10 minutes before the injection and continuing for about 60minutes. In some embodiments, the subject undergoes additional imageacquisition for about 10 minutes, or for about 20 minutes, or for about30 minutes, or for about 40 minutes, or for about 50 minutes, or forabout 60 minutes, at about one hour, or about two hours, or about 3hours, or about 4 hours, and at about 4 hours, or about 5 hours, orabout 6 hours, or about 7 hours, or about 8 hours, after the injectionof imaging agent-1.

In some embodiments, studies may also be performed using an agentspecialized for tissue blood flow using methods known to those familiarwith the art. The images from these studies may then be used todistinguish abnormalities seen in images from, for example, agent-1, dueto changes in NET from those due to alterations of global, regional orlocal blood flow.

Exemplary Cassettes and Reaction Systems

In some embodiments, systems, methods, kits, and cassettes are providedfor the synthesis of an imaging agent of the invention (e.g., imagingagent-1). In some embodiments, an imaging agent may be prepared using anautomated reaction system comprising a disposable or single usecassette. The cassette may comprise all the non-radioactive reagents,solvents, tubing, valves, reaction vessels, and other apparatus and/orcomponents necessary to carry out the preparation of a given batch ofimaging agent. The cassette allows the reaction system to have theflexibility to make a variety of different imaging agents with minimalrisk of cross-contamination, by simply changing the cassette. By theterm “cassette” is meant a piece of apparatus designed to fit removablyand interchangeably onto automated reaction systems, in such a way thatmechanical movement of moving parts of the automated reaction systemcontrols the operation of the cassette from outside the cassette, i.e.,externally. In certain embodiments, a cassette comprises a lineararrangement of valves, each linked to a port where various reagents,cartridges, syringes, and/or vials can be attached, by either needlepuncture of a septum-sealed vial, or by gas-tight, marrying joints. Eachvalve may have a male-female joint which interfaces with a correspondingmoving arm of the automated synthesizer. External rotation of the armcan control the opening or closing of the valve when the cassette isattached to the automated reaction system. Additional moving parts ofthe automated reaction system are designed to clip onto syringe plungertips, and thus raise or depress syringe barrels. An automated reactionsystem may further include a controller and one or more controllablevalves in electrical communication with the controller. An automatedreaction system may also include additional vessels, valves, sensors,heaters, pressurizing elements, etc., in electrical communication withthe controller. An automated reaction system may be operated by acontroller using suitable software for control of valve openings andclosings, heating, cooling, pressure levels, fluid movement, flow rate,etc. The automated reaction system may optionally include a computeroperating system, software, controls, etc., or other component. Inaddition, the automated reaction system may comprise a mount for thecassette.

Examples of automated reaction systems (e.g., a nucleophilic reactionsystem), include, but are not limited to, the Explora GN or RN synthesissystem (Siemens Medical Solutions USA, Inc.), GE-Tracerlab-MX synthesissystem (GE Healthcare), Eckert & Zeigler Modular-Lab Synthesis system,etc., which are commonly available at PET manufacturing facilities.

The automated reaction systems may carry-out numerous steps, as outlinedin FIG. 2, including, but not limited to, providing an ¹⁸F fluoridespecies, and an imaging agent precursor, optionally in a solution (e.g.,as described herein, for example, imaging agent precursor-1 inacetonitrile), a radiolabeling reaction (e.g., reaction of the ¹⁸Fspecies and the imaging agent precursor to form the imaging agent)optionally in a synthesis module, purification (e.g., by preparativeHPLC), solvent exchange (e.g., by SepPak), aseptic filtration, andrelease into a container.

In some embodiments, the automated reaction system may make use of acassette comprising a reaction module in fluid connection with apurification module and/or a formulation module. FIGS. 3 and 4 showschematic representations of cassettes in connection with exemplaryreaction systems for synthesizing an imaging agent comprising a reactionmodule, a purification module, and/or a formulation module. FIG. 5 showsschematic representation of an exemplary reaction system forsynthesizing an imaging agent comprising a reaction module. For example,the reaction module may include a reaction chamber in which conversionof the imaging agent precursor to the imaging agent is performed. Thereaction module may include a source of a fluoride species (e.g., ¹⁸F),a source of the imaging agent precursor, a source of a reagent (e.g.,salt), and other sources of additional components such as solvents, eachof which may optionally be fluidly connected to the reaction chamber.The reaction module may also comprise an anion exchange column forpurification of the fluoride species, prior to introduction into thereaction chamber.

Upon reaction, the resulting imaging agent product is transferred fromthe reaction module to the purification module for further processing,treatment, and/or purification. The purification module may include, forexample, a column (e.g., an HPLC column) fluidly connected to one ormore sources of solvents to be used as eluents. The purification modulemay further comprise a source of a stabilizing agent (e.g., ascorbicacid or a salt thereof), which may be added to the imaging agent uponpurification (e.g., by HPLC). The purified imaging agent is thentransferred to the formulation module, where further purification andformulation may be performed. The formulation module may include a C-18column for solvent exchange and/or a filter for aseptic filtration.

In another embodiment, a cassette comprises a reaction module and aformulation module. A reaction module of the invention may include asource of ¹⁸F, an anion exchange to remove unreacted [¹⁸O]H₂O, a sourceof an ammonium salt, a source for a diluent for the ¹⁸F, a source for animaging agent precursor, (e.g., imaging agent precursor-1 shown in FIG.1, or other imaging agent precursor), a source for an MeCN/H₂O diluentfor the reaction mixture, a reaction vessel for reacting the ¹⁸F and theimaging agent precursor, a solid phase extraction column (e.g., a C18column, or other suitable column) in fluid communication with thereaction vessel. The anion exchange column includes a solid sorbent toadsorb the ¹⁸F. Unreacted [¹⁸O]H₂O and residual reaction impurities passthrough cationic resin matrix without adsorbing on the sorbent. Thereaction module also includes a source of wash solutions in fluidcommunication with the anion exchange column for providing washsolutions to elute ¹⁸F off the sorbent, and includes a source of aneluent (e.g., as H₂O/MeCN, or other suitable eluent comprising a salt)in fluid communication with the anion exchange column for eluting theimaging agent product off the sorbent. The reaction module may alsoinclude a source of a diluent for the eluted ¹⁸F.

A formulation module of an apparatus of the invention may be in fluidcommunication with a reaction module and may include a solid phaseextraction cartridge that includes a solid sorbent (e.g., C-18, or othersuitable sorbent) to adsorb the diluted imaging agent, a source of washsolutions (e.g., comprising ascorbic acid, a salt thereof, or othersuitable wash solution) in fluid communication with the solid phaseextraction cartridge for providing wash solutions to wash off anyremaining impurities on the sorbent, and a source of eluting fluid(e.g., ethanol/H₂O, or other suitable eluting fluid) in fluidcommunication with the solid phase extraction cartridge for eluting theimaging agent product off the sorbent. The formulation module may alsoinclude a source of a diluent (e.g., comprising ascorbic acid, a saltthereof, or other suitable diluent), for diluting the eluted imagingagent. The formulation module may also be in fluid communication with asterilizing filter (e.g., a Millipore Millex GV PVDF sterilizing filter,or other suitable sterilizing filter).

In some embodiments, a general procedure for synthesizing an imagingagent of the invention (e.g., imaging agent-1) using an automatedsynthesis module is as follows. An [¹⁸F]-fluoride species (e.g., in anaqueous solution) is provided to a synthesis module. In some cases, thefluoride species (e.g., in an aqueous solution) is filtered through ananion exchange column to remove unreacted [¹⁸O]H₂O, wherein the[¹⁸F]-fluoride species is retained within the cationic resin matrix. Thecolumn is washed with solution (e.g., an aqueous base) to elute the[¹⁸F]-fluoride species into a reaction vessel. The resulting solution isdiluted (e.g., with MeCN), and then concentrated to dryness (e.g., usingelevated temperature and reduced pressure). The resulting material isexposed to solution of an imaging agent precursor (e.g., imaging agentprecursor-1), optionally in the presence on one or more reagents (e.g.,an activating agent). The solution is optionally heated for period oftime (e.g., to 90-110° C. and maintained 5-15 min), followed by cooling.The solution is evaporated to dryness (e.g., using elevated temperatureand/or reduced pressure), and then reconstituted in a reconstitutionsolution (e.g., H₂O/MeCN), followed by purification (e.g., by HPLC on anAgilent BONUS-RP column) using a select eluent (e.g., a solution ofNH₄HCO₂ in H₂O/MeCN). The product is collected, optionally diluted(e.g., with ascorbic acid solution), followed by transfer to aformulation module.

In a particular embodiment, a cassette is provided for use with anautomated synthesis module, for example, a GE TRACERlab MX synthesismodule. In one embodiment, a cassette comprises a disposable sterilizedassembly of molded stopcock manifolds specifically designed for use withthe automated synthesis module (e.g., GE TRACERlab MX synthesis module).Individual manifolds are connected in a linear or non-linear fashion toform a directional array that dictates the flow path of reagents used inthe preparation of an imaging agent (e.g., imaging agent-1). In someembodiments, the main body of the cassette contains at least onemanifold comprising a plurality of manifold positions (e.g., stopcocks).For example, the main body may comprise at least one, two, three, fouror more, manifolds. The cassette may comprise between 1 to 20 manifoldpositions, between 1 to 15 manifold positions, between 5 and 20 manifoldpositions, between 5 and 15 manifold positions. Each of the manifoldsmay or may not be symmetrical. In one embodiment, the main body of thecassette contains three plastic manifolds each fitted with five standardmolded stopcocks, thereby having a total of 15 total manifold positions.Individual stopcocks are adapted with luer fittings to accommodatesolvents, reagents, syringes, tubing required for gas and liquidhandling, etc. The stopcocks are adapted for solvents and reagents andmay be fitted with plastic spikes upon which inverted punch vials arelocated, while those featuring tubing and syringes are fitted with maleluer connections according to function. In some embodiments, thecassette comprises a linear arrangement of a plurality of stopcockmanifolds connected one or more of the components selected from thegroup consisting of a gas inlet, anion exchange cartridge, C-18cartridge, syringe, solvent reservoir, reaction vessel, HPLC system,collection vessel, reservoir for solution of ascorbic acid or saltthereof, and exhaust outlet. In some cases the cassette furthercomprises tubing. In some cases, the cassette further comprises animaging agent synthesis module, wherein the apparatus is fluidicallyconnected to the cassette. In some cases, the apparatus is capablecarrying out the method of synthesizing an imaging agent as describedherein (e.g., a method of synthesizing imaging agent-1).

A non-limiting example of a cassette configuration which may be used forthe preparation of imaging agent-1 is depicted in FIG. 3. The followingprovides a description of the attachments to each of the 15 manifoldpositions: 1) luer connections—gas inlet and [¹⁸O]H₂O recovery; 2) anionexchange cartridge—QMA Light; 3) spike connection—SWFI; 4)syringe-containing H₂O and/or MeCN; 5) luer connection—imaging agentprecursor-1; 6) luer connection—reaction vessel; 7) HPLC inlet; 8) luerconnection—ethanol; 9) luer connection—ascorbic acid; 10) luerconnection—collection vessel; 11) luer connection—final product vial;12) luer connection—tC18 light Sep Pak column inlet; 13) luerconnection—tC18 light Sep Pak column outlet; 14) syringe—containingascorbic acid; 15) luer connections—reaction vessel and exhaust.Manifold one (stopcocks 1-5) is joined to manifold two (stopcocks 6-10)and manifold two is connected to manifold three (stopcocks 11-15) usingtwo male luer connections fitted with a short length of silicon tubing.Individual manifold connections, luer fittings and all silicon tubingare readily available from commercial suppliers.

Another non-limiting example of a cassette configuration which may beused for the preparation of imaging agent-1 is depicted in FIG. 4. Thefollowing provides a description of the attachments to each of the 15manifold positions: 1) luer connections—gas inlet and [¹⁸O]H₂O recovery;2) anion exchange cartridge—QMA Light; 3) spike connection—MeCN; 4)syringe—empty; 5) spike connection—imaging agent precursor-1 (e.g., inMeCN); 6) luer connection—reaction vessel; 7) HPLC inlet; 8) spikeconnection—ascorbic acid; 9) luer connection—collection vessel; 10)syringe—containing ethanol and/or SFWI; 11) luer connection—finalproduct vial; 12) spike connection—H₂O and/or MeCN; 13) spikeconnection—ascorbic acid; 14) -syringe—empty; 15) luerconnections—reaction vessel and exhaust. Manifold one (stopcocks 1-5) isjoined to manifold two (stopcocks 6-10) using two male luer connectionsfitted with a short length of silicon tubing. Manifold two is connectedto manifold three (stopcocks 11-15) using a tC-18 Sep-Pak® and theappropriate luer adapters. Individual manifold connections, luerfittings and all silicon tubing are readily available from commercialsuppliers.

In some embodiments, the present invention provides a cassette for thepreparation of an imaging agent comprising the formula:

or a salt, free base, and/or pharmaceutically acceptable formula, orcombination thereof.

Pharmaceutical Compositions

Once a compound of the present disclosure (e.g., a compound of formula(I), (V), (VI), (VII), (IX) or (X))) has been prepared or obtained, itmay be combined with one or more pharmaceutically acceptable excipientsto form a pharmaceutical composition that is suitable for administrationto a subject, including a human. As would be appreciated by one of skillin this art, the excipients may be chosen, for example, based on theroute of administration as described below, the imaging agent beingdelivered, time course of delivery of the agent, and/or thehealth/condition of the subject. The pharmaceutical composition may be asolid or liquid.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

Pharmaceutically acceptable excipients include any and all solvents,diluents or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. General considerations informulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing the compound of the presentinvention (the “active ingredient”) into association with a carrierand/or one or more other accessory ingredients, and then, if necessaryand/or desirable, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and combinationsthereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium iodide, sodiummetabisulfite, sodium nitrite, sodium sulfite, and sodium thiosulfate.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as Cremophor,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionscan be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes can be used in the classical mantoux method of intradermaladministration.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

The pharmaceutical compositions of this invention can be administered tohumans and/or to animals parenterally (e.g., by intravenous,intramuscular, subcutaneous, or intraperitoneal injection). The mode ofadministration will vary depending on the intended use, as is well knownin the art.

Kits

Systems, methods, kits, and/or cassettes are provided comprising animaging agent or an imaging agent precursor as described herein or acomposition thereof and/or for preparation of an imaging agent (e.g.,imaging agent-1). In some embodiments, kits for the administration of animaging agent (e.g., imaging agent-1) are provided. In some cases, thecomposition provided with the kit may be used for or in the preparationof an imaging agent for detecting, imaging, and/or monitoring a disorderor condition. Kits of the invention may include, for example, acontainer comprising an imaging agent or an imaging agent precursor andinstructions for use. Kits may comprise a sterile, non-pyrogenic,formulation comprising a predetermined amount of an imaging agent or animaging agent precursor, and optionally other components. A containerthat may be used in conjunction with an imaging agent (e.g., imagingagent-1) for example, to deliver and/or administer the imaging agent toa subject, may be a syringe, bottle, vial, or tube. Instructions in akit of the invention may relate to methods for synthesizing an imagingagent or an imaging agent precursor, methods of diluting the imagingagent or the imaging agent precursor, methods of administering theimaging agent to a subject for diagnostic imaging, or other instructionsfor use. An imaging agent or an imaging agent precursor may be providedin a kit and additional preparations before use may optionally includediluting the imaging agent or imaging agent precursor to a usableconcentration.

In some cases, a kit can also include one or more vials containing adiluent for preparing an imaging agent (e.g., imaging agent-1)composition for administration to a subject (e.g., a human). A diluentvial may contain a diluent such as physiological saline or water, fordiluting imaging agent-1. For example imaging agent-1 may be packaged ina kit in a ready-to-inject formulation, or may require somereconstitution or dilution whereby a final composition/formulation forinjection or infusion is prepared.

Instructions in a kit of the invention may also include instructions foradministering the imaging agent to a subject and may include informationon dosing, timing, stress induction, etc. For example, a kit may includean imaging agent or imaging agent precursor as described herein alongwith instructions describing the intended application and the properadministration of the agent to a subject. As used herein, “instructions”can define a component of instruction and/or promotion, and typicallyinvolve written instructions on or associated with packaging of theinvention. Instructions also can include any oral or electronicinstructions provided in any manner such that a user will clearlyrecognize that the instructions are to be associated with the kit, forexample, audiovisual (e.g., videotape, DVD), internet, and/or web-basedcommunications. The written instructions may be in a form prescribed bya governmental agency regulating the manufacture, use or sale ofpharmaceuticals products, which instructions can also reflect approvalby the agency of manufacture, use; or sale for human administration. Insome cases, the instructions can include instructions for mixing aparticular amount of the diluent with a particular amount of aconcentrated solution of the imaging agent or a solid preparation of theimaging agent, whereby a final formulation for injection or infusion isprepared for example, such that the resulting solution is at a suitableconcentration for administration to a subject (e.g., at a concentrationas described herein). A kit may include a whole treatment regimen of theinventive compound.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing an agent described herein (e.g., an imagingagent precursor or an imaging agent). The agent may be in the form of aliquid, gel, or solid (e.g., powder). The agent may be preparedsterilely, packaged in a syringe, and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively, the kit may include an agent premixed and shipped in asyringe, vial, tube, or other container. The kit may have one or more orall of the components required to administer the agents to a subject,such as a syringe or i.v. needle tubing and bag.

It also will be understood that containers containing the components ofa kit of the invention, whether the container is a bottle, a vial (e.g.,with a septum), an ampoule, an infusion bag, or the like, can includeadditional indicia such as conventional markings that change color whenthe preparation has been autoclaved or otherwise sterilized. A kit ofthe invention may further include other components, such as syringes,labels, vials, tubing, catheters, needles, ports, and the like. In someaspect of the invention, a kit may include a single syringe containingthe imaging agent of the invention (e.g., imaging agent-1) sufficientfor administration and in some aspects of the invention a kit mayinclude more than one syringe.

Buffers useful in the preparation of imaging agents and kits include,for example, phosphate, citrate, sulfosalicylate, and acetate buffers. Amore complete list can be found in the United States Pharmacopoeia.Lyophilization aids useful in the preparation of imaging agents and kitsinclude, for example, mannitol, lactose, sorbitol, dextran, FICOLL®polymer, and polyvinylpyrrolidine (PVP). Stabilization aids useful inthe preparation of imaging agents and kits include, for example,ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodiummetabisulfite, gentisic acid, and inositol. Solubilization aids usefulin the preparation of imaging agents and kits include, for example,ethanol, glycerin, polyethylene glycol, propylene glycol,polyoxyethylene sorbitan monooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymers(e.g., Pluronics®) and lecithin. In certain embodiments, thesolubilizing aids are polyethylene glycol, cyclodextrins, and Pluronics.Bacteriostats useful in the preparation of imaging agents and kitsinclude, for example, benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl, or butyl paraben.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are listed here.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry,” Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

As used herein, the term “alkyl” is given its ordinary meaning in theart and refers to the radical of saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In some cases, the alkyl group may be a loweralkyl group, i.e., an alkyl group having 1 to 10 carbon atoms (e.g.,methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, ordecyl). In some embodiments, a straight chain or branched chain alkylmay have 30 or fewer carbon atoms in its backbone, and, in some cases,20 or fewer. In some embodiments, a straight chain or branched chainalkyl may have 12 or fewer carbon atoms in its backbone (e.g., C₁-C₁₂for straight chain, C₃-C₁₂ for branched chain), 6 or fewer, or 4 orfewer. Likewise, cycloalkyls may have from 3-10 carbon atoms in theirring structure, or 5, 6 or 7 carbons in the ring structure. Examples ofalkyl groups include, but are not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, hexyl, andcyclochexyl.

The terms “alkenyl” and “alkynyl” are given their ordinary meaning inthe art and refer to unsaturated aliphatic groups analogous in lengthand possible substitution to the alkyls described above, but thatcontain at least one double or triple bond respectively.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl,isobutyl, t-butyl, n-pentyl, sec-pentyl, isopentyl, t-pentyl, n-hexyl,sec-hexyl, moieties and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term “cycloalkyl,” as used herein, refers specifically to groupshaving three to ten, preferably three to seven carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic, or hetercyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “heteroalkyl” is given its ordinary meaning in the art andrefers to an alkyl group as described herein in which one or more carbonatoms is replaced by a heteroatom. Suitable heteroatoms include oxygen,sulfur, nitrogen, phosphorus, and the like. Examples of heteroalkylgroups include, but are not limited to, alkoxy, amino, thioester,poly(ethylene glycol), and alkyl-substituted amino

The terms “heteroalkenyl” and “heteroalkynyl” are given their ordinarymeaning in the art and refer to unsaturated aliphatic groups analogousin length and possible substitution to the heteroalkyls described above,but that contain at least one double or triple bond respectively.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CHF₂; —CH₂F; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alycyclic, heteroaliphatic, heterocyclic, aryl,heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic,heteroaliphatic, alkylaryl, or alkylheteroaryl substituents describedabove and herein may be substituted or unsubstituted, branched orunbranched, cyclic or acyclic, and wherein any of the aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments shown in the Examples thatare described herein.

The term “aryl” is given its ordinary meaning in the art and refers toaromatic carbocyclic groups, optionally substituted, having a singlering (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fusedrings in which at least one is aromatic (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl). That is,at least one ring may have a conjugated pi electron system, while other,adjoining rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls. The aryl group may be optionally substituted, asdescribed herein. Substituents include, but are not limited to, any ofthe previously mentioned substitutents, i.e., the substituents recitedfor aliphatic moieties, or for other moieties as disclosed herein,resulting in the formation of a stable compound. In some cases, an arylgroup is a stable mono- or polycyclic unsaturated moiety havingpreferably 3-14 carbon atoms, each of which may be substituted orunsubstituted. “Carbocyclic aryl groups” refer to aryl groups whereinthe ring atoms on the aromatic ring are carbon atoms. Carbocyclic arylgroups include monocyclic carbocyclic aryl groups and polycyclic orfused compounds (e.g., two or more adjacent ring atoms are common to twoadjoining rings) such as naphthyl groups.

The terms “heteroaryl” is given its ordinary meaning in the art andrefers to aryl groups comprising at least one heteroatom as a ring atom.A “heteroaryl” is a stable heterocyclic or polyheterocyclic unsaturatedmoiety having preferably 3-14 carbon atoms, each of which may besubstituted or unsubstituted. Substituents include, but are not limitedto, any of the previously mentioned substitutents, i.e., thesubstituents recited for aliphatic moieties, or for other moieties asdisclosed herein, resulting in the formation of a stable compound. Insome cases, a heteroaryl is a cyclic aromatic radical having from fiveto ten ring atoms of which one ring atom is selected from S, O, and N;zero, one, or two ring atoms are additional heteroatoms independentlyselected from S, O, and N; and the remaining ring atoms are carbon, theradical being joined to the rest of the molecule via any of the ringatoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and thelike.

It will also be appreciated that aryl and heteroaryl moieties, asdefined herein may be attached via an alkyl or heteroalkyl moiety andthus also include -(alkyl)aryl, -(heteroalkyl)aryl,-(heteroalkyl)heteroaryl, and -(heteroalkyl)heteroaryl moieties. Thus,as used herein, the phrases “aryl or heteroaryl moieties” and “aryl,heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)heteroaryl,and -(heteroalkyl)heteroaryl” are interchangeable. Substituents include,but are not limited to, any of the previously mentioned substituents,i.e., the substituents recited for aliphatic moieties, or for othermoieties as disclosed herein, resulting in the formation of a stablecompound.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one or more of the hydrogen atomsthereon independently with any one or more of the following moietiesincluding, but not limited to: aliphatic; alicyclic; heteroaliphatic;heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl;heteroalkylaryl; alkylheteroaryl; heteroalkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃;—CH₂F; —CHF₂; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x);—OCON(R_(x))₂; —N(R_(x))₂; —S(O)R_(x); —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alicyclic, heteroaliphatic, heterocyclic,aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl, heteroaryl,-(alkyl)aryl or -(alkyl)heteroaryl substituents described above andherein may be substituted or unsubstituted. Additionally, it will beappreciated, that any two adjacent groups taken together may represent a4, 5, 6, or 7-membered substituted or unsubstituted alicyclic orheterocyclic moiety. Additional examples of generally applicablesubstituents are illustrated by the specific embodiments describedherein.

The term “heterocycle” is given its ordinary meaning in the art andrefers to refer to cyclic groups containing at least one heteroatom as aring atom, in some cases, 1 to 3 heteroatoms as ring atoms, with theremainder of the ring atoms being carbon atoms. Suitable heteroatomsinclude oxygen, sulfur, nitrogen, phosphorus, and the like. In somecases, the heterocycle may be 3- to 10-membered ring structures or 3- to7-membered rings, whose ring structures include one to four heteroatoms.

The term “heterocycle” may include heteroaryl groups, saturatedheterocycles (e.g., cycloheteroalkyl) groups, or combinations thereof.The heterocycle may be a saturated molecule, or may comprise one or moredouble bonds. In some cases, the heterocycle is a nitrogen heterocycle,wherein at least one ring comprises at least one nitrogen ring atom. Theheterocycles may be fused to other rings to form a polycylicheterocycle. The heterocycle may also be fused to a spirocyclic group.In some cases, the heterocycle may be attached to a compound via anitrogen or a carbon atom in the ring.

Heterocycles include, for example, thiophene, benzothiophene,thianthrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, dihydropyrrole, pyrrolidine, imidazole,pyrazole, pyrazine, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, oxazine, piperidine, homopiperidine(hexamnethyleneimine), piperazine (e.g., N-methyl piperazine),morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, other saturated and/or unsaturated derivativesthereof, and the like. The heterocyclic ring can be optionallysubstituted at one or more positions with such substituents as describedherein. In some cases, the heterocycle may be bonded to a compound via aheteroatom ring atom (e.g., nitrogen). In some cases, the heterocyclemay be bonded to a compound via a carbon ring atom. In some cases, theheterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine,acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline,benzoquinoline, benzoisoquinoline, phenanthridine-1,9-diamine, or thelike.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “amino,” as used herein, refers to a primary (—NH₂), secondary(—NHR_(x)), tertiary (—NR_(x)R_(y)), or quaternary (—N⁺R_(x)R_(y)R_(z))amine, where R_(x), R_(y) and R_(z) are independently an aliphatic,alicyclic, heteroaliphatic, heterocyclic, aryl, or heteroaryl moiety, asdefined herein. Examples of amino groups include, but are not limitedto, methylamino, dimethylamino, ethylamino, diethylamino,methylethylamino, iso-propylamino, piperidino, trimethylamino, andpropylamino.

The term “alkyne” is given its ordinary meaning in the art and refers tobranched or unbranched unsaturated hydrocarbon groups containing atleast one triple bond. Non-limiting examples of alkynes includeacetylene, propyne, 1-butyne, 2-butyne, and the like. The alkyne groupmay be substituted and/or have one or more hydrogen atoms replaced witha functional group, such as a hydroxyl, halogen, alkoxy, and/or arylgroup.

The term “alkoxy” (or “alkyloxy”), or “thioalkyl” as used herein refersto an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom or through a sulfur atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-8 aliphaticcarbon atoms. In still other embodiments, the alkyl group contains 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains 1-4 aliphatic carbon atoms. Examples of alkoxy, include but arenot limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,t-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl include, butare not limited to, methylthio, ethylthio, propylthio, isopropylthio,n-butylthio, and the like.

The term “aryloxy” refers to the group, —O-aryl. The term “acyloxy”refers to the group, —O-acyl.

The term “alkoxyalkyl” refers to an alkyl group substituted with atleast one alkoxy group (e.g., one, two, three, or more, alkoxy groups).For example, an alkoxyalkyl group may be —(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl),optionally substituted. In some cases, the alkoxyalkyl group may beoptionally substituted with another alkyoxyalkyl group (e.g.,—(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl)-O—(C₁₋₆-alkyl), optionally substituted.

It will be appreciated that the above groups and/or compounds, asdescribed herein, may be optionally substituted with any number ofsubstituents or functional moieties. That is, any of the above groupsmay be optionally substituted. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds, “permissible” being in the context of the chemical rules ofvalence known to those of ordinary skill in the art. In general, theterm “substituted” whether preceded by the term “optionally” or not, andsubstituents contained in formulas of this invention, refer to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. It will be understood that “substituted”also includes that the substitution results in a stable compound, e.g.,which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc. In some cases,“substituted” may generally refer to replacement of a hydrogen with asubstituent as described herein. However, “substituted,” as used herein,does not encompass replacement and/or alteration of a key functionalgroup by which a molecule is identified, e.g., such that the“substituted” functional group becomes, through substitution, adifferent functional group. For example, a “substituted phenyl group”must still comprise the phenyl moiety and cannot be modified bysubstitution, in this definition, to become, e.g., a pyridine ring. In abroad aspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. Illustrative substituentsinclude, for example, those described herein. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valencies of the heteroatoms. Furthermore, this invention isnot intended to be limited in any manner by the permissible substituentsof organic compounds. Combinations of substituents and variablesenvisioned by this invention are preferably those that result in theformation of stable compounds useful for the formation of an imagingagent or an imaging agent precursor. The term “stable,” as used herein,preferably refers to compounds which possess stability sufficient toallow manufacture and which maintain the integrity of the compound for asufficient period of time to be detected and preferably for a sufficientperiod of time to be useful for the purposes detailed herein.

Examples of substituents include, but are not limited to, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl,heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, halide,alkylthio, oxo, acylalkyl, carboxy esters, -carboxamido, acyloxy,aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl,arylamino, aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl,-carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-,aminocarboxamidoalkyl-, cyano, alkoxyalkyl, perhaloalkyl,arylalkyloxyalkyl, and the like.

As used herein, the term “determining” generally refers to the analysisof a species or signal, for example, quantitatively or qualitatively,and/or the detection of the presence or absence of the species orsignals.

The term “diagnostic imaging,” as used herein, refers to a procedureused to detect an imaging agent.

The term “diagnosis” as used herein encompasses identification,confirmation, and/or characterization of a condition, a disease, and/ora disorder.

A “diagnostic kit” or “kit” comprises a collection of components, termedthe formulation, in one or more vials which are used by the practicingend user in a clinical or pharmacy setting to synthesize diagnosticradiopharmaceuticals. For example, the kit may be used by the practicingend user in a clinical or pharmacy setting to synthesize and/or usediagnostic radiopharmaceuticals. In some embodiments, the kit mayprovide all the requisite components to synthesize and use thediagnostic pharmaceutical except those that are commonly available tothe practicing end user, such as water or saline for injection and/orthe radioisotope (e.g., ¹⁸F). equipment for processing the kit duringthe synthesis and manipulation of the radiopharmaceutical, if required,equipment necessary for administering the radiopharmaceutical to thesubject such as syringes, shielding, imaging equipment, and the like. Insome embodiments, imaging agents may be provided to the end user intheir final form in a formulation contained typically in one vial orsyringe, as either a lyophilized solid or an aqueous solution.

As used herein, a “portion of a subject” refers to a particular regionof a subject, location of the subject. For example, a portion of asubject may be the brain, heart, vasculature, cardiac vessels, etc., ofa subject.

As used herein a “session” of testing may be a single testing protocolthat a subject undergoes.

As used herein, the term “subject” refers to a human or non-human mammalor animal. Non-human mammals include livestock animals, companionanimals, laboratory animals, and non-human primates. Non-human subjectsalso specifically include, without limitation, horses, cows, pigs,goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, mink, andrabbits. In some embodiments of the invention, a subject is referred toas a “patient.” In some embodiments, a patient or subject may be underthe care of a physician or other health care worker, including, but notlimited to, someone who has consulted with, received advice from orreceived a prescription or other recommendation from a physician orother health care worker.

Any of the compounds described herein may be in a variety of forms, suchas, but not limited to, salts, solvates, hydrates, tautomers, andisomers.

In certain embodiments, the imaging agent is a pharmaceuticallyacceptable salt of the imaging agent. The term “pharmaceuticallyacceptable salt” as used herein refers to those salts which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al., describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 1977, 66, 1-19, incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄-alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counter ions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

In certain embodiments, the compound is in the form of a hydrate orsolvate. The term “hydrate” as used herein refers to a compoundnon-covalently associated with one or more molecules of water. Likewise,the term “solvate” refers to a compound non-covalently associated withone or more molecules of an organic solvent.

In certain embodiments, the compound described herein may exist invarious tautomeric forms. The term “tautomer” as used herein includestwo or more interconvertable compounds resulting from at least oneformal migration of a hydrogen atom and at least one change in valency(e.g., a single bond to a double bond, a triple bond to a single bond,or vice versa). The exact ratio of the tautomers depends on severalfactors, including temperature, solvent, and pH. Tautomerizations (i.e.,the reaction providing a tautomeric pair) may be catalyzed by acid orbase. Exemplary tautomerizations include keto-to-enol; amide-to-imide;lactam-to-lactim; enamine-to-imine; and enamine-to-(a different) enaminetautomerizations.

In certain embodiments, the compounds described herein may exist invarious isomeric forms. The term “isomer” as used herein includes anyand all geometric isomers and stereoisomers (e.g., enantiomers,diasteromers, etc.). For example, “isomer” includes cis- andtrans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixturesthereof, as falling within the scope of the invention. For instance, anisomer/enantiomer may, in some embodiments, be provided substantiallyfree of the corresponding enantiomer, and may also be referred to as“optically enriched.” “Optically-enriched,” as used herein, means thatthe compound is made up of a significantly greater proportion of oneenantiomer. In certain embodiments the compound of the present inventionis made up of at least about 90% by weight of a preferred enantiomer. Inother embodiments the compound is made up of at least about 95%, 98%, or99% by weight of a preferred enantiomer. Preferred enantiomers may beisolated from racemic mixtures by any method known to those skilled inthe art, including chiral high pressure liquid chromatography (HPLC) andthe formation and crystallization of chiral salts or prepared byasymmetric syntheses. See, for example, Jacques, et al., Enantiomers,Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972).

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Synthesis of3-(4-((1,2-Bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propyl4-methylbenzenesulfonate

Example 1A Synthesis of1,2-bis(tert-butoxycarbonyl)-1-[3-bromo-4-(3-hydroxypropoxy)benzyl]-guanidine

To a solution of1,2-bis(tert-butoxycarbonyl)-1-[3-bromo-4-hydroxybenzyl]-guanidine (forsynthesis, see, for example, Purohit et al., International PCT PatentPublication No. WO2008/083056, incorporated herein by reference) (2.0 g,4.51 mmol) dissolved in anhydrous DMF (45 mL) was added K₂CO₃ (1.12 g,8.13 mmol), and 3-bromopropanol (816 mg, 5.87 mmol) and the reactionmixture warmed to 50° C. using an oil bath. After 2 h, the reactionmixture was diluted with water (30 mL), and the aqueous layer separatedthen extracted with EtOAc (3×100 mL). The combined organic layers weredried over MgSO₄, filtered and concentrated to a solid. The crudematerial was purified using silica gel chromatography (4:1 to 3:2hexanes:EtOAc) to yield a white solid product (2.00 g, 88% yield). ¹HNMR (CDCl₃, 600 MHz): δ 9.42 (brs, 1H), 9.27 (brs, 1H), 7.54 (d, J=1.8Hz, 1H), 7.26 (m, 1H), 6.85 (d, J=2.4 Hz, 1H), 5.08 (brs, 2H), 4.19 (t,J=5.4 Hz, 2H), 3.92 (m, 2H), 2.16 (m, 1H), 2.18 (m, 2H), 1.51 (s, 9H),1.43 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz): δ 163.8, 160.8, 155.0, 154.3,144.8, 133.1, 132.6, 127.9, 113.0, 111.7, 84.7, 79.2, 67.8, 60.6, 46.7,31.9, 28.5, 28.3.

Example 1B Synthesis of3-(4-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propyl4-methylbenzenesulfonate

To a solution of the product of Example 1A (339 mg, 0.676 mmol)dissolved in anhydrous CH₂Cl₂ (6.76 mL) was added TsCl (155 mg, 0.812mmol), DMAP (99 mg, 0.812 mmol) and Et₃N (0.141 mL, 1.01 mmol). Thereaction mixture was stirred at room temperature for 1.5 h thenconcentrated to a yellow oil. The crude material was directly purifiedusing silica gel chromatography (4:1 hexanes:EtOAc) to yield a colorlessoil (384.3 mg, 87% yield). ¹H NMR (CDCl₃, 600 MHz): δ 7.74 (d, J=8.4 Hz,2H), 7.50 (d, J=1.8 Hz, 1H), 7.21 (m, 3H), 6.70 (d, J=8.4 Hz, 1H), 5.08(brs, 2H), 4.30 (t, J=6.0 Hz, 2H), 4.00 (t, J=6.0 Hz, 2H), 2.37 (s, 3H),2.16 (m, 2H), 1.51 (s, 9H), 1.43 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz): δ160.6, 154.9, 154.0, 145.0, 133.0, 132.9, 132.7, 130.0, 128.0, 112.9,111.9, 84.7, 79.0, 67.0, 64.1, 46.4, 29.0, 28.5, 28.2, 21.8.

Example 2 Synthesis of3-(4-((1,2-Bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propyl4-bromobenzenesulfonate

To a solution of the product of Example 1A (300 mg, 0.598 mmol)dissolved in anhydrous CH₂Cl₂ (6.0 mL) was added BsCl (183.3 mg, 0.718mmol), DMAP (87.7 mg, 0.718 mmol) and Et₃N (0.125 mL, 0.897 mmol). Thereaction mixture was stirred at room temperature for 2.5 h thenconcentrated to an oil. The crude material was directly purified usingsilica gel chromatography (4:1 hexanes:EtOAc) to yield a colorless oil(395.6 mg, 92% yield). ¹H NMR (CDCl₃, 300 MHz): δ 9.40 (brs, 2H),7.72-7.67 (m, 2H), 7.55-7.50 (m, 3H), 7.24 (dd, J=3, 9 Hz, 1H), 6.69 (d,J=9 Hz, 1H), 5.11 (brs, 2H), 4.35 (t, J=6.0 Hz, 2H), 3.97 (t, J=6.0 Hz,2H), 2.18 (m, 2H), 1.47 (s, 9H), 1.39 (s, 9H); ¹³C NMR (4:1,CDCl₃:DMSO-d₆, 150 MHz): δ 160.7, 160.5, 157.1, 153.5, 134.0, 132.0,131.6, 130.3, 130.2, 128.6, 128.3, 127.2, 127.2, 112.4, 111.3, 84.5,79.0, 66.8, 63.4, 42.3, 27.4.

Example 3 Synthesis of3-(4-((1,2-Bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propylmethanesulfonate

To a solution of the product of Example 1A (300 mg, 0.598 mmol)dissolved in anhydrous CH₂Cl₂ (6.0 mL) was added MsCl (55.8 μL, 0.718mmol), DMAP (87.7 mg, 0.718 mmol) and Et₃N (0.125 mL, 0.897 mmol). Thereaction mixture was stirred at room temperature for 45 min thenconcentrated to yield an oil. The crude material was directly purifiedusing silica gel chromatography (4:1 hexanes:EtOAc) to yield a colorlessoil (245.6 mg, 71% yield). ¹H NMR (CDCl₃, 300 MHz): δ 9.35 (brs, 2H),7.56 (d, J=3.0 Hz, 1H), 7.26 (m, 1H). 6.84 (d, J=9.0 Hz, 1H), 5.09 (brs,2H), 4.53 (t, J=6.0 Hz, 2H), 4.15 (t, J=6.0 Hz, 2H), 3.01 (s, 3H), 2.29(m, 2H), 1.52 (s, 9H), 1.43 (s, 9H); ¹³C (CDCl₃, 150 MHz): δ 160.7,154.9, 154.1, 133.3, 133.1, 128.0, 132.2, 113.2, 110.7, 128.3, 84.7,80.5, 66.9, 64.6, 46.7, 29.9, 28.5, 28.2.

Example 4 Synthesis of3-(4-((1,2-Bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propyltrifluoromethanesulfonate

To a solution of the product of Example 1A (300 mg, 0.598 mmol)dissolved in anhydrous CH₂Cl₂ (6.0 mL) was added Tf₂O (203 mg, 0.718mmol), DMAP (87.7 mg, 0.718 mmol) and Et₃N (0.125 mL, 0.897 mmol). Thereaction mixture was stirred at room temperature for 1.5 h thenconcentrated to yield an oil. The crude material was directly purifiedusing silica gel chromatography (4:1 to 1:1 hexanes:EtOAc) to yield acolorless oil (312 mg, 82% yield). ¹H NMR (CDCl₃, 300 MHz): δ 9.39 (brs,2H), 7.54 (d, J=3.0 Hz, 1H), 7.26 (m, 1H), 6.84 (d, J=9.0 Hz, 1H), 5.08(brs, 2H), 4.16 (t, J=6.0 Hz, 2H), 3.81 (t, J=6.0 Hz, 2H), 2.27 (m, 2H),1.50 (s, 9H), 1.39 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz): δ 160.7, 154.9,154.3, 133.2, 132.8, 128.1, 113.2, 112.0, 84.7, 79.3, 65.8, 46.7, 40.7,32.4, 28.5, 28.2; ¹⁹F NMR (CDCl₃, 282 MHz): δ −75.5 (s).

Example 5

The following Example describes the synthesis of compounds of Formula(II), including but not limited to imaging agent precursor-1. TheExample more specifically provides the synthesis of the trifluoroaceticacid salt of imaging agent precursor-1, according to the scheme shown inFIG. 6.

Example 5A Synthesis of 3-bromo-4-(3-hydroxypropoxy)benzonitrile(Compound 1)

3-Bromo-4-hydroxy benzonitrile (10.0 g, 50.5 mmol) was dissolved inacetone and successively treated with 1-bromo-3-propanol (19.0 g, 138mmol) and K₂CO₃ (20.9 g, 151 mmol) at ambient temperature. The resultingsuspension was warmed to 50° C. and maintained 3 d. After cooling toambient temperature, the solids were removed by filtration, exhaustivelywas with acetone and the filtrate concentrated. Purification bychromatography on SiO₂ (A: hexanes; B: EtOAc; 0-100% B over 35.4 min;200 mL/min; 330 g column) afforded a solid. Further purification byrecrystallization from hot MTBE (131 mL) and pentane (130 mL), withcooling at −20° C. (12 h) to induce precipitation, afforded a solid (7.2g, 58%). ¹H NMR (300 MHz, CDCl₃) δ 7.78 (s, 1H), 7.56 (d, J=9 Hz, 1H),6.94 (d, J=6 Hz, 1H), 4.23 (t, J=6 Hz, 2H), 3.88 (t, J=6 Hz, 2H), 2.08(m, J=6 Hz, 2H).

Example 5A-1

The following Example describes the synthesis of Compound 1, using analternate synthetic method to Example 5A. 3-Bromo-4-hydroxybenzonitrile(0.100 kg, 0.505 mol) was added to a reaction vessel followed by2-butanone (1.00 L), 3-chloro-1-propanol (50 mL, 0.598 mol), Na₂CO₃(80.6 g, 0.760 mol), and NaI (15.0 g, 0.100 mol). The reaction mixturewas then shielded from light using aluminum foil, heated to reflux andstirred overnight. After 23 h, unreacted starting material remained.Additional 3-chloro-1-propanol (8.7 mL, 0.10 mol) was then added, andthe mixture returned to reflux. After 34 h total reflux time, the heatwas removed, and the vessel cooled slowly over 19 h to 22.8° C. beforeaddition of MTBE (1.00 L). The resulting solution was stirred 44 minthen filtered through a class C sintered glass funnel containing a 5 cmCelite bed. The reaction vessel and Celite bed were rinsed with severalsmall portions of MTBE, and the combined filtrates concentrated invacuo.

The crude solid was dissolved in refluxing MTBE (410 mL) then treatedwith heptane (410 mL) over 14 min to form an oil. Upon completion of theaddition, the heating mantle was removed and the biphase cooled to 29.9°C. After 1 h, the resulting suspension was diluted with heptane (1.18L), stirred 66 min then filtered through a class C sintered glassfunnel. The solids were washed with 9:1 heptane:MTBE (398 mL) thentransferred to a drying pan and placed in a vacuum oven. After drying at35±5° C. for 36 h, 118.4 g of the solid was obtained (0.462 mol; 91.5%).

Example 5B Synthesis of 3-bromo-4-(3-hydroxypropoxy) benzylaminehydrochloride (Compound 2)

Compound 1 (5.0 g, 19.5 mmol) was suspended in THF then stirred atambient temperature until complete dissolution was observed. BH₃.THF(42.9 mmol; 42.9 mL of a 1.0 M solution in THF) was then added dropwiseand the resulting mixture heated to reflux. After 5 h, the mixture wascooled to 4° C. then carefully treated with MeOH (50 mL). HCl(g) wasbubbled through the solution for 30 min then all volatiles removed invacuo. The white solid thus obtained was dissolved in MeOH (17.8 mL)then successively treated with MTBE (36 mL) and hexanes (40 mL). Theresulting suspension was stirred 30 min, the white solids collected thendried to constant weight (4.7 g, 81%). This material was used directlyin the subsequent step without further purification.

Example 5B-1

The following Example describes the synthesis of Compound 2, using analternate synthetic method to Example 5B. Compound 1 (118.4 g, 0.462mol) was transferred, under nitrogen, to a reaction vessel along withanhydrous THF (1.16 L). The mixture was stirred until completedissolution was observed then slowly treated with BH₃.THF (1.02 mol;1.02 L of a 1.0 M solution in THF) over 20 min. Following completeaddition, the reaction vessel was heated to reflux and maintainedovernight. The resulting suspension was then cooled to 29.9° C. beforean ice water bath was applied to further reduce the internal temperatureto 4.9° C. Hydrochloric acid (1.25 mol; 1.00 L of 1.25 M solution inMeOH) was then added dropwise over 94 min; a measured value of pH 3confirmed complete hydrolysis of the intermediate boronate species. Theresulting mixture was then concentrated to dryness in vacuo (<35° C.) toyield a solid (172.1 g).

The crude product was transferred to a new, clean reaction vessel alongwith MeOH (279 mL). After stirring 20 min, the resulting suspension wastreated with MTBE (550 mL), stirred 16 min then diluted with heptane(1.10 L). After 2.5 h, the solids were isolated by filtration through aclass C sintered glass funnel then washed with 1:1 heptane:MTBE (410 mL)before transfer to a vacuum oven. After drying at 35±5° C. for 10 h,119.2 g of a solid material was obtained.

Example 5C Synthesis of1,3-bis(tert-butoxycarbonyl)-[3-bromo-4-(3-hydroxypropoxy)benzyl]-guanidine(Compound 3)

Compound 2 (0.438 g, 1.48 mmol) was dissolved in MeOH (7.00 mL), andsuccessively treated with N,N′-bis-tert-butoxycarbonyl-1H-pyrazolecarboxamidine (0.412 g, 1.33 mmol) and i-Pr₂NEt (0.380 g, 2.95 mmol) atambient temperature. The resulting mixture was stirred 3 h thenconcentrated and purified by chromatography on SiO₂ (A: hexanes; B:EtOAc; 0-100% B over 19.2 min; 40 mL/min; 40 g column) to obtain theproduct as a white foam (0.61 g, 82%). ¹H NMR (300 MHz, CDCl₃) δ 8.5 (t,1H), 7.5 (d, 1H), 7.2 (dd, 1H), 6.85 (d, 1H), 4.52 (d, 2H), 4.18 (t,2H), 3.9 (t, 2H), 2.1 (m, 2H), 1.52 (s, 9H), 1.47s (s, 9H).

Example 5C-1

The following Example describes the synthesis of Compound 3, using analternate synthetic method to Example 5C. Compound 2 (119.1 g, 0.401mol) was transferred to a reaction vessel with MeOH (1.13 L),N,N′-bis-tert-butoxycarbonyl-1H-pyrazole carboxamidine (126.4 g, 0.408mol), and i-Pr₂NEt (82.0 ml, 0.461 mol). The resulting mixture wasstirred at ambient temperature for 13 h then treated with EtOAc (150 mL)and concentrated to dryness in vacuo (305.7 g). The crude oil thusobtained was transferred to a separatory funnel using 1.31 L of EtOActhen washed with deionized water (417 mL). The aqueous layer was furtherwashed with EtOAc (600 mL), and the combined organic layers successivelywashed with 307 mL 0.5 M NaHSO₄—H₂O, 300 mL deionized water and 300 mL0.5 M NaHCO₃ then dried over excess Na₂SO₄. The drying agent was removedby filtration through a class C sintered glass funnel then washed withEtOAc (190 mL). The combined filtrates were concentrated in vacuo toyield a light brown, viscous oil (213 g).

Example 5D Synthesis of3-(4-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propyl4-bromobenzenesulfonate (Compound 4)

Compound 3 (0.2 g, 0.4 mmol) was successively treated with4-bromobenzenesulfonyl chloride (173 mg, 0.677 mmol), Et₃N (80.62 mg,0.796 mmol), DMAP (4.86 mg, 3.98 μmol) and CH₂Cl₂ (8 mL) at ambienttemperature. The resulting solution was stirred 24 h then all volatilesremoved in vacuo. The residue was triturated with hexanes:EtOAc (10 mL;9:1 v/v) to obtain a white solid which was collected by filtration.Purification by chromatography on SiO₂ (A: hexanes; B: EtOAc; 0-100% Bover 15.4 min; 35 ml/min; 24 g column) afforded the product as a stickywhite solid (174 mg, 60%). 1H NMR (300 MHz, CDCl₃) δ 8.53 (t, 1H), 7.66(m, 2H), 7.5 (m, 3H), 7.17 (m, 1H), 6.67 (d, J=8.4 Hz, 1H), 4.53 (d, J=5Hz, 2H), 4.33 (t, J=6 Hz, 2H), 3.93 (t, J=6 Hz, 2H), 2.16 (m, 2H), 1.53(s, 9H), 1.47 (s, 9H).

Example 5D-1

The following Example describes the synthesis of Compound 4, using analternate synthetic method relative to Example 5D. Compound 3 (212.9 g,0.424) was transferred to a reaction vessel, under nitrogen, usinganhydrous CH₂Cl₂ (2.00 L) then stirred 15 min until complete dissolutionoccurred. The resulting solution was successively treated with4-bromobenzenesulfonyl chloride (125.5 g, 0.491 mol), Et₃N (80.0 mL,0.573 mol) and DMAP (2.06 g, 0.017 mol) then vigorously stirred 16 h atambient temperature. NOTE: the process was relatively exothermic as theinternal temperature reached 33.9° C. following addition of the DMAP.Additional 4-bromobenzenesulfonyl chloride (10.5 g, 0.041 mol) was thenadded and the resulting mixture stirred 19 h. This process was repeatedonce again using additional 4-bromobenzenesulfonyl chloride (20.9 g,0.082 mol) and Et₃N (11.3 mL, 0.081 mol) followed by 8 h of vigorousstirring at ambient temperature. The resulting solution was then treatedwith deionized water (600 mL) with transfer to a separatory funnel. Thelayers were then separated and the aqueous layer was washed with CH₂Cl₂(290 mL). The combined organic layers were further washed with 5%aqueous NaHCO₃ (380 mL), dried over an excess of Na₂SO₄, then filteredand concentrated in vacuo. The crude product was partially purified bysilica gel chromatography (˜20 g SiO₂/g of crude product) using 10-20%EtOAc/heptane; like fractions were combined and concentrated to a solidin vacuo. The crude material thus obtained was further purified throughtrituration from MTBE (804 mL) and heptane (1580 mL) then isolated byfiltration through a class C sintered glass funnel. The filter cake waswashed with 9:1 heptane/MTBE (467 mL) then transferred to a vacuum ovenand dried 15 h at ambient temperature (149.4 g, 0.207 mol; 48.9%).

Example 5E Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, trifluoroacetate salt (TFA salt of ImagingAgent Precursor-1)

A 25 mL round bottom flask was charged with Compound 4 (3.00 g, 4.15mmol) then CH₂Cl₂ (6 mL), and the resulting suspension stirred untilcomplete dissolution was observed. Trifluoroacetic acid (6 mL, 78.3mmol) was then added and the mixture stirred an additional 4 h. Allvolatiles were then removed, and the residue treated with EtOAc (20 mL).The resulting mixture was stirred at room temperature for 3 h, duringwhich time a white solid precipitated. The solids were collected on asintered glass funnel of medium porosity then exhaustively washed withEtOAc (20 mL) and dried to constant weight (2.5 g, 95%). 1H NMR (400MHz, DMSO-d₆) δ 7.54 (m, 4H), 7.4 (d, 1H), 7.15 (m, 1H), 6.9 (d, 1H),4.15 (m, 4H), 3.86 (m, 2H), 1.92 (m, 2H).

Example 5E-1

The following Example describes the synthesis of the TFA salt of imagingagent precursor-1, using an alternate synthetic method relative toExample 5E. Compound 4 (149.4 g, 0.207 mol) was dissolved in CH₂Cl₂(1.20 L) then treated with TFA (300 mL) in one portion at ambienttemperature. After 14 h, all volatiles were removed in vacuo and thecrude oil directly treated with EtOAc (1.32 L). After 3 h, the resultingsuspension was filtered through a class C sintered glass funnel and thesolids washed with EtOAc (2×140 mL). The filter cake was thentransferred to a glass drying pan and placed in a vacuum oven for 12 hat ambient temperature.

Example 6 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, hydrochloric acid salt

A 25 mL round bottom flask was charged with Compound 4 (2.00 g, 2.77mmol) then HCl (28.0 mmol; 7.00 mL of a 4.0 M solution in dioxane), andthe resulting solution stirred 4 h. The white solid thus obtained wascollected, exhaustively washed with MTBE (20 mL) then dried to constantweight (1.4 g, 2.51 mmol; 90.6%). ¹H NMR (400 MHz, D₂O+DMSO-d₆) δ 6.94(d, 2H), 6.76 (d, 2H), 6.74 (s, 1H), 6.45 (m, 1H), 6.17 (d, 1H), 3.53(m, 4H), 3.15 (t, 2H), 1.36 (m, 2H), 0.5 (s, 1H).

Example 7 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, p-toluenesulfonic acid salt

A 25 mL round bottom flask was charged with Compound 4 (0.50 g, 0.69mmol), p-toluenesulfonic acid hydrate (1.32 g, 6.93 mmol) and THF (6mL). The resulting solution was heated to reflux under a nitrogenatmosphere, maintained 6 h then slowly cooled to ambient temperatureovernight. The white solid precipitate thus obtained was collected,exhaustively washed with Et₂O and dried to a constant weight (0.328 g,0.473 mmol; 68.3%). ¹H NMR (300 MHz, DMSO-d₆) δ 7.74 (m, 5H), 7.48 (d,1H), 7.45 (m, 2H), 7.23 (dd, J=3 Hz, 1H), 7.08 (m, 3H), 6.99 (d, J=9 Hz,1H), 4.25 (m, J=6 Hz, 3H), 3.97 (t, J=6 Hz, 2H), 2.26 (s, 3H), 2.04 (m,2H).

Example 8 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, acetic acid salt

The product of Example 6 (200 mg, 0.359 mmol) was dissolved in THF/H₂O(2 mL; 1:1 v/v) then treated with AgOAc (3 mL of a 22 mg/mL solution in1:4 MeCN/H₂O); immediate precipitation was observed. The slurry wasstirred 20 min then filtered through a 0.45 μm PVDF filter disc, and thefiltrate lyophilized. The amorphous salt thus obtained was dissolved inCH₂Cl₂ (1 mL), stirred 2 h ambient temperature then cooled to 5° C. andmaintained 3 h. The resulting white crystalline solids were collected byfiltration then air dried (0.100 g, 0.172 mmol; 48.0%). ¹H NMR (400 MHz,DMSO-d₆) δ 9.6 (brs, 1H), 7.77 (m, 4H), 7.49 (d, 1H), 7.2 (m, 1H), 7.0(d, 1H), 4.26 (m, 4H), 3.97 (t, 2H), 2.06 (m, 2H), 1.66 (s, 3H)

Example 9 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, benzoic acid salt

The product of Example 6 (415 mg, 0.744 mmol) was dissolved in THF/H₂O(4.2 mL; 1:1 v/v) then treated with AgOBz (10 mL of a 16 mg/mL solutionin 1:4 MeCN/H₂O); immediate precipitation was observed. The slurry wasstirred 20 min then filtered through a 0.45 m PVDF filter disc and thefiltrate lyophilized. The amorphous salt thus obtained was dissolved inEtOAc (10 mL), stirred 2 h ambient temperature then cooled to 5° C. andmaintained 3 h. The resulting white crystalline solids were collected byfiltration, washed with EtOAc (1 mL) then air dried (0.090 g, 0.140mmol; 18.8%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.27 (brs, 1H), 7.88 (brs,3H), 7.76 (m, 4H), 7.52 (d, 2H), 7.31 (m, 4H), 7.0 (d, 1H), 4.26 (m,4H), 3.97 (m, 2H), 2.06 (m, 2H), 1.66 (s, 3H)

Example 10 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, phosphoric acid salt

Compound 4 (0.200 g, 0.277 mmol) was dissolved in CH₂Cl₂/TFA (2 mL, 4:1v/v) then stirred overnight at ambient temperature. All volatiles werethen removed in vacuo, and the resulting thick oil further dried in avacuum oven (2 h at 25° C. and 5 mbar). EtOAc (2 mL) and phosphoric acid(0.30 mmol; 62 μL of 5M solution in THF) were then added, and theresulting mixture refluxed 3-5 min. After cooling to ambienttemperature, MTBE (1 mL) was added. The resulting suspension wasfiltered through a sintered glass funnel, air dried then placed in avacuum oven (48 h at 25° C. and 5 mbar; 0.164 g, 2.65 mmol; 96.2%). 1HNMR (400 MHz, D₂O+DMSO-d₆) δ 7.92 (q, 4H), 7.55 (s, 1H), 7.35 (m, 1H),7.05 (d, 1H), 4.33 (m, 4H), 4.03 (t, 2H), 2.13 (m, 2H), 1.25 (s, 1.5H).

Example 11 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, methanesulfonic acid salt

Compound 4 (1.00 g, 1.38 mmol) was dissolved in CH₂Cl₂ (8 mL) thentreated with distilled TFA (2 mL) dropwise at ambient temperature andstirred overnight. All volatiles were removed and the residuesuccessively treated with EtOAc (10 mL) and MsOH (1.52 mmol; 153 μL of a10 M solution in THF). The resulting solution was heated to reflux,maintained 3-5 min then slowly cooled to ambient temperature in the oilbath. The solid product was isolated by filtration, air dried thenplaced in a vacuum oven (48 h at 25° C. and 5 mbar; 0.838 g, 1.36 mmol;98.6%). ¹H NMR (400 MHz, D₂O+DMSO-d₆) δ 7.75 (d, 4H), 7.5 (s, 1H), 7.26(d, 1H), 7.0 (d, 1H), 4.31 (m, 4H), 3.95 (t, 2H), 2.33 (s, 3H), 2.07 (m,2H).

Example 12 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, sulfuric acid salt

Compound 4 (0.100 g, 0.157 mmol) was suspended in dioxane (0.5 mL) thentreated with sulfuric acid (0.158 mmol; 158 μL of 1 M solution in THF)at ambient temperature; additional dioxane (400 μL) was required forcomplete dissolution. The resulting solution was shaken several minutesthen concentrated in vacuo (overnight at 25° C. and 5 mbar). The crudesolid mass was triturated with hot EtOAc, briefly sonicated and cooledprior to filtration. The resulting solid material was further dried invacuo to obtain the final product. ¹H NMR (400 MHz, D₂O+DMSO-d₆) δ 9.8(s, 1H), 8.1 (s, 1H), 7.75 (q, 4H), 7.5 (d, 1H), 7.25 (brs and m, 4H),7.0 (d, 1H), 4.25 (m, 4H), 3.95 (t, 2H), 2.0 (m, 2H).

Example 13 Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-methylbenzenesulfonate, trifluoroacetate salt (TFA salt of ImagingAgent Precursor-2)

Example 13A Synthesis of3-(4-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-2-bromophenoxy)propyl4-methylbenzenesulfonate

A round bottom flask was successively charged with Compound 3 (2.00 g,3.98 mmol), 4-toluenesulfonyl chloride (0.987 g, 5.17 mmol), Et₃N (0.604g, 5.97 mmol), DMAP (0.139 g, 1.19 mmol), and CH₂Cl₂ (16 mL) at ambienttemperature. After 5 h, the reaction mixture was poured into aseparatory funnel, washed with water (10 mL) and brine (10 mL) thendried over MgSO₄, filtered, and concentrated to a foam. The solid wasredissolved in CH₂Cl₂ (4 mL) then loaded onto a 40 g silica column(Redisep R_(f)) and purified using a Teledyne ISCO Combiflash instrument(A: hexanes; B: EtOAc; 0-100% B over 19.2 min; 40 mL/min) to obtain theproduct as a white solid (1.89 g, 72.3%). ¹H NMR (300 MHz, CDCl₃) δ11.54 (s, 1H), 8.56 (brt, 1H), 7.75 (d, 2H, J=4.5 Hz), 7.47 (d, 1H, J=3Hz), 7.22 (m, 3H), 6.75 (d, 1H, J=4.5 Hz), 4.55 (d, 2H, J=6 Hz), 4.32(t, 2H, J=6 Hz), 3.98 (t, 2H, J=6 Hz), 2.36 (s, 3H), 2.16 (m, 2H), 1.54(s, 9H), 1.50 (s, 9H).

Example 13B Synthesis of 3-(2-Bromo-4-(guanidinomethyl)phenoxy)propyl4-bromobenzenesulfonate, trifluoroacetate salt (TFA salt of ImagingAgent Precursor-2)

A round bottom flask was charged with the product of Example 13A (1.50g, 2.28 mmol) then treated with a solution of TFA in CH₂Cl₂ at ambienttemperature (52 mmol: 1:1 v/v, 8 mL). After 3.5 h, the mixture wasconcentrated to a thick oil then treated with acetone (2 mL) andconcentrated once again. The acetone evaporation process was repeatedtwo additional times, and the residue thus obtained was dissolved inCH₂Cl₂ (4 mL). The CH₂Cl₂ was again removed in vacuo, and the processrepeated two additional times to obtain the crude product as a paleyellow solid. The solid was finally washed with MTBE (2×10 mL) and EtOAc(5 mL) to yield the TFA salt of imaging agent precursor-2 as a freeflowing white powder. ¹H NMR (300 MHz, DMSO-d₆) δ 7.98 (t, 1H, J=6 Hz),7.74 (d, 2H, J=9 Hz), 7.51 (d, 1H, J=3 Hz), 7.34 (d, 2H, J=9 Hz), 7.26(dd, 1H, J=3, 9 Hz), 7.02 (d, 1H, J=9 Hz), 4.30 (d, 2H, J=6 Hz), 4.22(t, 2H, J=6 Hz), 3.99 (t, 2H, J=6 Hz), 2.35 (s, 3H), 2.07 (m, 2H).

Example 14 Salt Stability Study

The long term chemical integrity of various salt forms of imaging agentprecursor-1 were evaluated though monitoring the weight percent purityof solid samples aged under controlled storage conditions: 40 and 70° C.and 60% relative humidity. The data shown in FIG. 7 and tabulated inTable 1 detail some of the observed differences.

Example 15 Physical Properties of Selected Salt Forms

Selected physical properties of the salts of Examples 5-6 and 8-12,determined using established characterization methods, are tabulatedbelow (Table 1).

TABLE 1 Summary of physical properties of salt forms HydrochlorideMesylate Phosphate Sulfate Acetate Benzoate TrifluoroacetateCrystallinity Crystalline Crystalline Crystalline CrystallineCrystalline Crystalline Crystalline Stoichiometry 1 equiv 1 equiv 1equiv 1 equiv 1 equiv 1 equiv 1 equiv Hygroscopicity Indicates hydrateSlight Slight Hygroscopic -NA- -NA- Slight formation hygroscopicityhygroscopicity hygroscopicity Stability to GVS and Stable Stable StableMixture of -NA- -NA- Stable 40° C./75% RH phases Thermal First event atFirst event at First event at First event at -NA   -NA   First event atstability 117° C. 152° C. 155° C. 103° C. 142° C. Solubility -NA- 1.280.3 -NA- 0.4 1.5 4.68 (Acetonitrile; mg/mL)

The following Examples (16-20) detail development of the combination ofsteps used for the manufacture of imaging agent-1. A flow chart of theoverall process is shown in FIG. 2.

Example 16 Preparation of [¹⁸F]fluoride

[¹⁸F]Fluoride was produced by proton bombardment of [¹⁸O]H₂O in acyclotron; the nuclear chemical transformation is shown below and may besummarized as ¹⁸O(p,n)¹⁸F. For purposes of the bombardment, the chemicalform of the ¹⁸O is H₂ ¹⁸O. The chemical form of the resulting ¹⁸F isfluoride ion.

¹⁸O+proton→¹⁸F+neutron

According to established industry procedures, [¹⁸O]H₂O (2-3 mL) housedwithin a tantalum target body using Havar® foil, was bombarded with 11MeV protons (nominal energy); where the proton threshold energy for thereaction is 2.57 MeV and the energy of maximum cross section is 5 MeV.Target volume, bombardment time and proton energy each may be adjustedto manage the quantity of [¹⁸F]fluoride produced.

Example 17 Synthesis of1-{3-Bromo-4-[3-[¹⁸F]fluoropropoxy]benzyl}guanidine (Imaging Agent-1)

The product of Example 16 was transferred from cyclotron to thesynthesis module, then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with aqueous K₂CO₃with transfer to the reaction vessel. The resulting solution was dilutedwith MeCN then concentrated to dryness using elevated temperature andreduced pressure. The anhydrous [¹⁸F]KF thus obtained was individuallytreated with MeCN solutions of the products of Example 5E, 7 or 11 andKryptofix® 222 then warmed to 110° C. and maintained 15 min.

Example 17A Synthesis of1-{3-Bromo-4-[3-[¹⁸F]fluoropropoxy]benzyl}guanidine, formic acid salt(Formic acid salt of imaging agent-1)

Example 17B Development of Preparative HPLC Purification Method

Selection of parameters suitable for purification of the product ofExample 17 was achieved through detailed study of the chromatographicbehavior of the product of various salts of imaging agent precursor-1.Initial column screening was performed using a 9.5%/min gradient from5-95% MeCN containing 0.1% HCO₂H and 10% H₂O at 1.00 mL/min, whichrevealed improved specificity over known impurities when using theAgilent Zorbax BONUS-RP (4.6×150 mm) column; selected chromatograms areprovided in FIG. 8.

Following column selection, a detailed study of the optimal solventmodifier was conducted, where the counterion, concentration and ionicstrength were adjusted to balance compound resolution and retention. Asummary of the experimental parameters evaluated are tabulated below(Table 2).

TABLE 2 Summary of HPLC Purification - TFA salt of imaging agentprecursor-1 using Agilent Zorbax BONUS-RP Concentration ionic RetentionTailing Modifier (mM) pH strength (min) Factor HCO₂H 22 2.81 — 6.88 0.91HCO₂NH₄ 10 3.13 0.001 6.74 0.89 HCO₂NH₄ 10 3.97 0.006 7.45 1.08 HCO₂NH₄10 4.5 0.008 7.71 1.14 HCO₂NH₄ 5 4.5 0.004 7.76 1.18 HCO₂NH₄ 15 4.50.012 7.91 1.24 MeCO₂NH₄ 10 4.03 0.001 6.77 0.82 MeCO₂NH₄ 10 4.46 0.0037.34 1.04 MeCO₂NH₄ 10 5.48 0.008 8.01 1.32

Example 17C Synthesis of1-{3-Bromo-4-[3-[¹⁸F]fluoropropoxy]benzyl}guanidine, formic acid salt

The product of Example 17 was cooled to ambient temperature and thesolution concentrated. The crude product was diluted with H₂O/MeCN (1mL, 4:1 v/v) then directly purified by HPLC on an Agilent ZorbaxBONUS-RP column using a solution of NH₄HCO₂ in H₂O/MeCN. The mainproduct peak was collected then assayed to determine radiochemical yieldand purity.

TABLE 3 Summary of radiochemical yield and purity from various precursorsalt forms Imaging agent precursor-1 Example 7 Example 11 RadiochemicalYield 60%  35% 15% Radiochemical Purity 99% 100% 99%

Example 18 General Preparation of Imaging Agent-1

The following Example describes a general procedure for synthesizingimaging agent-1, using an automated synthesis module. Aqueous[¹⁸F]fluoride, as prepared in Example 16, was transferred from thecyclotron to a synthesis module, then filtered through an anion exchangecolumn to remove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained withinthe cationic resin matrix. The column was then washed with aqueous basewith transfer to the reaction vessel. The resulting solution wasoptionally diluted with MeCN then concentrated to dryness using elevatedtemperature and reduced pressure. The mixture of anhydrous [¹⁸F]fluorideand base thus obtained was treated with a solution of imaging agentprecursor-1 (or a salt thereof), optionally an activating agent thenwarmed to 90-110° C. and maintained 5-15 min. After cooling, thesolution was evaporated to dryness using elevated temperature andreduced pressure then reconstituted in H₂O/MeCN and directly purified byHPLC on an Agilent BONUS-RP column using a solution of NH₄HCO₂ inH₂O/MeCN. The main product peak was collected, diluted with ascorbicacid then transferred to the formulation module.

Example 18A-1 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe Eckert & Ziegler Modular-Lab Synthesis Module

The product of Example 15 was transferred from a cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with K₂CO₃ (11.5 μmol;0.500 mL of a 23.0 mM solution in H₂O) with transfer to the reactionvessel. The resulting solution was diluted with MeCN (0.500 mL) thenconcentrated to dryness using a two step procedure; heating to 135° C.for 5 min under vacuum and nitrogen flow (500 mL/min) then at 100° C.for 10 min under vacuum and nitrogen flow (500 mL/min). The mixture ofanhydrous [¹⁸F]KF and K₂CO₃ thus obtained was treated with a solution ofthe TFA salt of imaging agent precursor-1 5.00 mg, 7.87 μmol) andKryptofix® 222 (22.5 mg, 59.7 μmol) in t-BuOH:MeCN (4:1 v/v; 1.5 mL)then warmed to 110° C. and maintained 15 min. The resulting solution wascooled to 95° C. then concentrated for 5 min under a flow of nitrogen.The mixture was then treated with H₂O/MeCN (4:1 v/v; 1.00 mL) and warmedto 100° C. for 5 min. After cooling for 60 sec, the resulting solutionwas directly purified by HPLC on an Agilent BONUS-RP (10 μm; 9.4×250 mm)column using a 82:18 H₂O/MeCN eluent containing NH₄HCO₂ (pH 3.8) at aflow rate of 5 mL/min. The main product peak eluting at 12-14 min wascollected, diluted with ascorbic acid (10 mL of a 0.28 M solution inH₂O; pH 4) then transferred to the formulation module; 50% decaycorrected radiochemical yield.

Example 18A-2 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe Eckert & Ziegler Modular-Lab Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with K₂CO₃ (2.01 μmol;0.500 mL of a 4.02 mM solution in H₂O) with transfer to the reactionvessel. The resulting solution was diluted with MeCN (0.500 mL) thenconcentrated to dryness using a two step procedure; heating to 135° C.for 3 min under vacuum and nitrogen flow (500 mL/min) then at 100° C.for 9 min under vacuum and nitrogen flow (500 mL/min). The mixture ofanhydrous [¹⁸F]KF and K₂CO₃ thus obtained was treated with a solution ofthe product of Example 7 (1.00 mg, 1.44 μmol) and Kryptofix® 222 (4.11mg, 11.0 μmol) in t-BuOH:MeCN (4:1 v/v; 1.5 mL) then warmed to 110° C.and maintained 15 min. The resulting solution was cooled to 95° C. thenconcentrated for 5 min under a flow of nitrogen. The mixture was thentreated with H₂O/MeCN (4:1 v/v; 1.00 mL) and warmed to 100° C. for 5min. After cooling for 60 sec, the resulting solution was directlypurified by HPLC on an Agilent BONUS-RP (10 μm; 9.4×250 mm) column usinga 82:18 H₂O/MeCN eluent containing NH₄HCO₂ (pH 3.8) at a flow rate of 5mL/min. The main product peak eluting at 12-14 min was collected,diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂O; pH 4)then transferred to the formulation module; 33% decay correctedradiochemical yield.

Example 18A-3 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe Eckert & Ziegler Modular-Lab Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with K₂CO₃ (2.01 μmol;0.500 mL of a 4.02 mM solution in H₂O) with transfer to the reactionvessel. The resulting solution was diluted with MeCN (0.500 mL) thenconcentrated to dryness using a two step procedure; heating to 135° C.for 3 min under vacuum and nitrogen flow (500 mL/min) then at 100° C.for 9 min under vacuum and nitrogen flow (500 mL/min). The mixture ofanhydrous [¹⁸F]KF and K₂CO₃ thus obtained was treated with a solution ofthe product of Example 11 (0.88 mg, 1.44 μmol) and Kryptofix® 222 (4.11mg, 11.0 μmol) in t-BuOH:MeCN (4:1 v/v; 1.5 mL) then warmed to 110° C.for 15 min. The resulting solution was cooled to 95° C. thenconcentrated for 5 min under a flow of nitrogen. The mixture was thentreated with H₂O/MeCN (4:1 v/v; 1.00 mL) and warmed to 100° C. for 5min. After cooling for 60 sec, the resulting solution was directlypurified by HPLC on an Agilent BONUS-RP (10 μm; 9.4×250 mm) column usinga 82:18 H₂O/MeCN eluent containing NH₄HCO₂ (pH 3.8) at a flow rate of 5mL/min. The main product peak eluting at 12-14 min was collected,diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂O; pH 4)then transferred to the formulation module; 15% decay correctedradiochemical yield.

Example 18A-4 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe Eckert & Ziegler Modular-Lab Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with Et₄NHCO₃ (39.4μmol; 0.500 mL of a 78.8 mM solution in H₂O) with transfer to thereaction vessel. The resulting solution was diluted with MeCN (0.500 mL)then concentrated to dryness using a two step procedure; heating to 135°C. for 5 min under vacuum and nitrogen flow (500 mL/min) then at 100° C.for 10 min under vacuum and nitrogen flow (500 mL/min). The mixture ofanhydrous [¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with asolution of imaging agent precursor-1 (5.00 mg, 7.87 μmol) int-BuOH:MeCN (4:1 v/v; 1.0 mL) then warmed to 110° C. for 15 min. Theresulting solution was cooled to 95° C. then concentrated for 5 minunder a flow of nitrogen. The mixture was then treated with H₂O/MeCN(4:1 v/v; 1.00 mL) and warmed to 100° C. for 5 min. After cooling for 60sec, the resulting solution was directly purified by HPLC on an AgilentBONUS-RP (10 μm; 9.4×250 mm) column using a 82:18 H₂O/MeCN eluentcontaining NH₄HCO₂ (pH 3.8) at a flow rate of 5 mL/min. The main productpeak eluting at 12-14 min was collected, diluted with ascorbic acid (10mL of a 0.28 M solution in H₂O; pH 4) then transferred to theformulation module; 46% decay corrected radiochemical yield.

Example 18A-5 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe Eckert & Ziegler Modular-Lab Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with Et₄NHCO₃ (31.5μmol; 0.500 mL of a 63.0 mM solution in H₂O) with transfer to thereaction vessel. The resulting solution was diluted with MeCN (0.500 mL)then concentrated to dryness using a two step procedure; heating to 135°C. for 5 min under vacuum and nitrogen flow (500 mL/min) then at 100° C.for 10 min under vacuum and nitrogen flow (500 mL/min). The mixture ofanhydrous [¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with asolution of the TFA salt of imaging agent precursor-1 (4.00 mg, 6.30μmol) in MeCN (1.0 mL) then warmed to 110° C. for 15 min. The resultingsolution was cooled to 95° C. then concentrated for 5 min under a flowof nitrogen. The mixture was then treated with H₂O/MeCN (4:1 v/v; 1.00mL) and warmed to 100° C. for 5 min. After cooling for 60 sec, theresulting solution was directly purified by HPLC on an Agilent BONUS-RP(10 μm; 9.4×250 mm) column using a 82:18 H₂O/MeCN eluent containingNH₄HCO₂ (pH 3.8) at a flow rate of 5 mL/min. The main product peakeluting at 12-14 min was collected, diluted with ascorbic acid (10 mL ofa 0.28 M solution in H₂O; pH 4) then transferred to the formulationmodule; 42% decay corrected radiochemical yield.

Example 18A-6 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe Eckert & Ziegler Modular-Lab Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with Et₄NHCO₃ (39.5μmol; 0.500 mL of a 79.0 mM solution in H₂O) with transfer to thereaction vessel. The resulting solution was diluted with MeCN (0.500 mL)then concentrated to dryness using a two step procedure; heating to 135°C. for 5 min under vacuum and nitrogen flow (500 mL/min) then at 100° C.for 10 min under vacuum and nitrogen flow (500 mL/min). The mixture ofanhydrous [¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained was treated with asolution of the TFA salt of imaging agent precursor-2 (4.50 mg, 7.87μmol) in MeCN (1.0 mL) then warmed to 110° C. and maintained 15 min. Theresulting solution was cooled to 95° C. then concentrated for 5 minunder a flow of nitrogen. The mixture was then treated with H₂O/MeCN(4:1 v/v; 1.00 mL), warmed to 100° C. and maintained 5 min. Aftercooling 60 sec, the resulting solution was directly purified by HPLC onan Agilent BONUS-RP (10 μm; 9.4×250 mm) column using a 82:18 H₂O/MeCNeluent containing NH₄HCO₂ (pH 3.8) at a flow rate of 5 mL/min. The mainproduct peak eluting at 12-14 min was collected, diluted with ascorbicacid (10 mL of a 0.28 M solution in H₂O; pH 4) then transferred to theformulation module; 46% decay corrected radiochemical yield.

Example 18B-1 Preparation of Formic Acid Salt of Imaging Agent-1 Usingthe GE TRACERLab MX Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with K₂CO₃ (11.5 μmol;0.800 mL of a 14.4 mM solution in H₂O) with transfer to the reactionvessel. The resulting solution was then concentrated to dryness using atwo step procedure; heating to 95° C. for 3 min under vacuum andnitrogen flow then at 115° C. for 7 min under vacuum and nitrogen flow.The mixture of anhydrous [¹⁸F]KF and K₂CO₃ thus obtained was treatedwith a solution of the TFA salt of imaging agent precursor-1 (5.00 mg,7.87 μmol) and Kryptofix® 222 (22.5 mg, 59.7 μmol) in t-BuOH:MeCN (4:1v/v; 1.5 mL) then warmed to 110° C. and maintained 15 min. The resultingsolution was cooled to 95° C. then concentrated for 7 min under a flowof nitrogen. The mixture was then treated with H₂O/MeCN (4:1 v/v; 5.00mL) and then warmed to 95° C. for 5 min. After cooling to 50° C., theresulting solution was directly purified by HPLC on an Agilent BONUS-RP(10 μm; 9.4×250 mm) column using a 82:18 H₂O/MeCN eluent containingNH₄HCO₂ (pH 3.8) at a flow rate of 5 mL/min. The main product peakeluting at 10-12 min was collected, diluted with ascorbic acid (10 mL ofa 0.28 M solution in H₂O; pH 4), and then transferred to the formulationmodule; 20% decay corrected radiochemical yield. A flow diagram for theprocess outlined above is provided in FIG. 3.

Example 18B-2 Preparation of Formic Acid Salt of Imaging Agent-1 usingthe GE TRACERLab MX Synthesis Module

The product of Example 16 is transferred from cyclotron to the synthesismodule then filtered through an anion exchange column to removeunreacted [¹⁸O]H₂O; [¹⁸F]fluoride is retained within the cationic resinmatrix. The column is then washed with Et₄NHCO₃ (39.5 μmol; 0.500 mL ofa 79.0 mM solution in H₂O) with transfer to the reaction vessel. Theresulting solution is then concentrated to dryness using a two stepprocedure; heating to 95° C. for 3 min under vacuum and nitrogen flowthen at 115° C. for 7 min under vacuum and nitrogen flow. The mixture ofanhydrous [¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained is treated with asolution of the TFA salt of imaging agent precursor-2 (4.50 mg, 7.87μmol) in MeCN (1.0 mL) then warmed to 90° C. and maintained 10 min. Theresulting solution is cooled to 95° C. then concentrated for 7 min undera flow of nitrogen. The mixture is then treated with H₂O/MeCN (4:1 v/v;2.00 mL), warmed to 90° C. and maintained 5 min. After cooling to 50°C., the resulting solution is directly purified by HPLC on an AgilentBONUS-RP (10 μm; 9.4×250 mm) column using a 82:18 H₂O/MeCN eluentcontaining NH₄HCO₂ (pH 3.8) at a flow rate of 5 mL/min. The main productpeak eluting at 10-12 min is collected, diluted with ascorbic acid (10mL of a 0.28 M solution in H₂O; pH 4) then transferred to theformulation module. A flow diagram for the process outlined above isprovided in FIG. 4.

Example 18C Preparation of Formic Acid Salt of Imaging Agent-1 using theGE TRACERLab FX Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with K₂CO₃ (11.5 μmol;0.800 mL of a 14.4 mM solution in H₂O) with transfer to the reactionvessel. The resulting solution was then concentrated to dryness using atwo step procedure; heating to 68° C. for 3 min under vacuum and heliumflow then at 95° C. for 4 min under vacuum and helium flow. The mixtureof anhydrous [¹⁸F]KF and K₂CO₃ thus obtained was cooled to 70° C.,treated with a solution of the TFA salt of imaging agent precursor-1(5.00 mg, 7.87 μmol) and Kryptofix® 222 (22.5 mg, 59.7 μmol) int-BuOH:MeCN (4:1 v/v; 1.5 mL) then warmed to 95° C. and maintained 15min. The resulting solution was cooled to 55° C. then concentrated for 7min under a flow of helium. The mixture was further treated with H₂O(0.1 mL), maintained 2 min then cooled to 40° C. and diluted withH₂O/MeCN (4:1 v/v; 3.00 mL). The resulting solution was directlypurified by HPLC on an Agilent BONUS-RP (10 μm; 9.4×250 mm) column usinga 82:18 H₂O/MeCN eluent containing NH₄HCO₂ (pH 3.8) at a flow rate of 5mL/min. The main product peak eluting at 9-11 min was collected, dilutedwith ascorbic acid (10 mL of a 0.28 M solution in H₂O; pH 4) thentransferred to the formulation module; 40% decay corrected radiochemicalyield.

Example 18D Preparation of Formic Acid Salt of Imaging Agent-1 using theSiemens Explora RN Synthesis Module

The product of Example 16 was transferred from cyclotron to thesynthesis module then filtered through an anion exchange column toremove unreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within thecationic resin matrix. The column was then washed with K₂CO₃ (11.5 μmol;0.800 mL of a 14.4 mM solution in H₂O) with transfer to the reactionvessel. The resulting solution was then concentrated to dryness using atwo step procedure; heating to 95° C. for 2 min under vacuum andnitrogen flow then at 115° C. for 5 min under vacuum and nitrogen flow.The mixture of anhydrous [¹⁸F]KF and K₂CO₃ thus obtained wassuccessively treated with a solution of the TFA salt of imaging agentprecursor-1 (4.00 mg, 6.30 μmol) in MeCN (1.00 mL) and Kryptofix® 222(18.0 mg, 47.8 μmol) also in MeCN (0.50 mL) then warmed to 110° C. andmaintained 15 min. The resulting solution was cooled to 95° C. thenconcentrated for 5 min under a flow of nitrogen. The mixture was thencooled to 55° C., treated with H₂O/MeCN (4:1 v/v; 1.00 mL) and directlypurified by HPLC on an Agilent BONUS-RP (10 μm; 9.4×250 mm) column usinga 82:18 H₂O/MeCN eluent containing NH₄HCO₂ (pH 3.8) at a flow rate of 5mL/min. The main product peak eluting at 12-14 min was collected,diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂O; pH 4)then transferred to the formulation module; 32% decay correctedradiochemical yield.

Example 18E Preparation of Formic Acid Salt of Imaging Agent-1 using theExplora GN Synthesis Module

The product of Example 16 is transferred from cyclotron to the synthesismodule then filtered through an anion exchange column to removeunreacted [¹⁸O]H₂O; [¹⁸F]fluoride was retained within the cationic resinmatrix. The column is then washed with Et₄NHCO₃ (39.5 μmol; 1.00 mL of a39.5 mM solution in H₂O) with transfer to the reaction vessel. Theresulting solution is diluted with MeCN (1.00 mL) then concentrated todryness; 110-115° C. Additional MeCN (1.50 mL) is then added and thesolution concentrated to dryness once again. The mixture of anhydrous[¹⁸F]Et₄NF and Et₄NHCO₃ thus obtained is treated with a solution of theTFA salt of imaging agent precursor-2 (4.50 mg, 7.87 μmol) in MeCN (1.0mL) then warmed to 90° C. and maintained 10 min. The resulting solutionis cooled to 60° C. then concentrated to dryness; 95° C. The mixture isthen treated with H₂O/MeCN (4:1 v/v; 2.00 mL), warmed to 100° C. andmaintained 5 min. After cooling to 60° C., the resulting solution isdirectly purified by HPLC on an Agilent BONUS-RP (10 μm; 9.4×250 mm)column using a 82:18 H₂O/MeCN eluent containing NH₄HCO₂ (pH 3.8) at aflow rate of 5 mL/min. The main product peak eluting at 12-14 min iscollected, diluted with ascorbic acid (10 mL of a 0.28 M solution inH₂O; pH 4) then transferred to the formulation module. A flow diagramfor the process outlined above is provided in FIG. 5.

Example 19 Solvent Exchange

Imaging agent-1 was transferred from purification to the formulationmodule then filtered through a tC18 Sep-Pak® cartridge to remove MeCN;imaging agent-1 was retained on the C18 matrix, and the filtrate wasdiscarded. The cartridge was successively washed with ascorbic acid (10mL of a 0.28 M solution in H₂O; pH 4), the filtrate discarded, thenEtOH/H₂O (1.00 mL; 1:1 v/v), and the filtrate collected. The ethanolconcentrate thus obtained was further diluted with ascorbic acid (9.0 mLof a 0.28 M solution in H₂O; pH 5.8) in preparation for final asepticfiltration.

Example 20 Aseptic Filtration Process

The final product vial assembly was constructed from the followingpre-sterilized components: one 30 mL product vial, one Millipore MillexGV4 venting filter (0.22 μm×4 mm), one tuberculin syringe (1 mL) and oneinsulin syringe (0.5 mL). The product of Example 19 was then transferredfrom formulation to the final product vial assembly through a MilliporeMillex GV PVDF sterilizing filter (0.22 μm×13 mm). Quality controlsamples are then removed, using the syringe assemblies, to complete allproduct release requirements.

Example 21

Evaluation of several experimental parameters in the nucleophilicfluorination of imaging agent precursor-1 using the K₂CO₃/Kryptofix® 222reagent combination initially revealed that while overall reactioncomplexity increased with added K₂CO₃, fluorination efficiency remainedunchanged above 0.66 molar equivalents (FIG. 9A). Elevated base (e.g.,carbonate) levels were primarily correlated to unproductive consumptionof starting material (e.g., imaging agent precursor-1), with hydrolysisto the derived alcohol as the primary decomposition pathway. Severalalternate base combinations (Table 4), including modification of thepotassium counterion as well as substitution of organic amine bases,proved less effective as promoters of the fluorination reaction (<10%conversion).

TABLE 4 Comparison of base identity and fluorination yield. Base % yieldK₂CO₃ 45-60 KHSO₄ <10 K₂HPO₄ <10 KH₂PO₄ <10 i-Pr₂NEt <10Tetramethylguanidine <10 Pyridine <10

Lower fluorination yield was also observed in the absence of Kryptofix®222, regardless of K₂CO₃ stoichiometry. However, the presence ofKryptofix® 222 markedly increased solution pH (10-12).

The fluorination reaction was also evaluated in several solvent systems,including MeCN, t-BuOH, and mixtures thereof; DMF, DMSO, and THF alone.MeCN and t-BuOH:MeCN combinations proved the most effective. Analysis ofcrude reaction mixtures from each solvent combination revealed aspecific impurity profile resulting from unproductive consumption ofimaging agent precursor-1 (FIG. 9B). MeCN alone provided the bestcombination of fluorination efficiency and overall impurity profile.

A subsequent series of studies revealed that both release of ¹⁸F fromthe anion exchange column and fluorination efficiency are markedlyinfluenced by the identity, concentration, and composition of the basicsolution utilized during transfer of ¹⁸F from the cyclotron to thereaction vessel. Specifically, we noted that regardless of cationidentity (e.g., potassium or tetraalkylammonium such astetraethylammonium or tetrabutylammonium), there exists a thresholdconcentration of the anionic solution component (HO⁻, HCO₃ ⁻, MsO⁻,TsO⁻, I⁻), below which a decreased efficiency of ¹⁸F release occurred.Notably however, effective release of ¹⁸F was not necessarily associatedwith efficient fluorination. Within the tetrabutylammonium series alone(Table 5), we determined that while the bicarbonate anion was superiorto other aniond, reaction efficiency was less than with the combinationof K₂CO₃/Kryptofix® 222 described above (e.g., Tables 4-5). Increasingbicarbonate concentration improved overall fluorination efficiency. FIG.9C shows the effect of concentration of tetraalkylammonium bicarbonateutilized for anion exchange on fluorination efficiency. The combinationof five molar equivalents of Et₄NHCO₃ and either imaging agentprecursor-1 or -2 provided equal fluorination efficiency as well as animproved overall impurity profile compared to the originalK₂CO₃/Kryptofix® 222 system.

TABLE 5 Comparison of tetrabutylammonium salt form and fluorinationyield. Salt form % yield Mesylate <2 Hydroxide <5 Tosylate <10 Iodide18.7 Bicarbonate (8.8 mM) 28.0 Bicarbonate (34.7 mM) 60.0

The non-radioactive experiments outlined above were adapted for themanufacture of imaging agent-1 on both the Siemens Explora RN and Eckert& Ziegler ModularLab remote synthesis modules. Multivariate screeningstudies (base, time and temperature) on the individual modules thusprovided the unit-specific parameters required to maintain chemicalfidelity across discrete instruments; specific parameters are describedin Example 18.

Example 22

A human study was performed that determined the quantification of normalpattern of regional myocardial radioactivity concentration of imagingagent-1.

Methods:

Normal subjects (n=6) were injected with ˜220 MBq of imaging agent-1intravenously, and dynamic PET images were acquired over 80 min withoutpatient movement. Attenuation corrected images were re-oriented intostandard cardiac specific axes, and the maximal regional myocardialuptake was quantified on a sector-by-sector basis using WLCQ software.The hearts were divided into three short axis slices (Base-B; Mid-M;Apical-A) and four radial sectors (Anterior-A; Septal-S; Inferior-I;Lateral-L) and mean regional uptake for each sector was calculated.Activity was expressed as Bq/ml.

Results:

The radiotracer cleared quickly from the blood and demonstrated afavorable biodistribution for early cardiac imaging. Regional and globalmyocardial activity peaked within the first 10 min and reached a plateauat ˜60 min post injection. There was no significant variation (p=0.69,ANOVA) in regional myocardial uptake at this time around thecircumference of the heart (A: 11592±2474 Bq/ml; S: 11647±2829 Bq/ml; I:11818±1991 Bq/ml; L:11424±2439 Bq/ml). There was also no significant(p=0.08, ANOVA) base-to-apex gradient in myocardial uptake (B:11284±2844Bq/ml; M:11898±2047 Bq/ml; A:11678±2148 Bq/ml).

The myocardial radioactivity concentration of the imaging agent-1 wasuniform throughout the heart in normal volunteers. This studyestablished the normal pattern of quantitative regional myocardialradioactivity concentration. This type of regional myocardial analysisprovides advantages over evaluation of heart-to-mediastinal ratios infuture studies of patients with heart disease.

Example 23

Dosimetry in non-human primates of imaging agent-1 was examined. Imagingagent-1, which is labeled with ¹⁸F, is a novel norepinephrinetransporter (NET) ligand and was a useful radiotracer for mapping thecardiac nerve terminal in vivo using positron emission tomography. Astudy was performed in four non-human primates to estimate humanradiation dosimetry.

Methods:

In this study two male and two female cynomolgus monkeys were imagedusing a Concord Focus 220 MicroPET scanner for whole body ¹⁸Fdistribution following 4 to 5 mCi (0.65 to 1.6 μg) single intravenousinjection of imaging agent-1. Under isoflurane anesthesia, images of theanimals from head to lower abdomen were acquired in 5 segments over fourand half hours following injection. Radioactivity in identifiable organsand the remainder of the body was determined as a function of time usingregion-of-interest analysis. The total number of disintegrations perunit injected dose was determined by normalizing by the injectedradioactivity and integrating over time the data for radioactivityversus time. Using the OLINDA/EXM software (Organ Level Internal DoseAssessment/EXponential Modeling Software, published by VanderbiltUniversity), the normalized number of ¹⁸F disintegrations for each organwas combined with the energy released by each disintegration, and usingthe MIRD schema, estimates were made of the fraction of the totalreleased energy that was retained in each source organ and thecontribution from each source organ to the energy deposited insurrounding target organs for an adult human. Dividing the totalfractional energy deposited in each organ by the corresponding organmass yielded the radiation dose for that organ per unit (mCi or MBq)injected dose.

Results:

From the radiation dose estimates, it was predicted that the human organthat would receive the highest dose was the urinary bladder wall with anaverage of 0.41±0.089 rem/mCi. The next five highest-dose organs andtheir respective mean dose estimates were the kidneys (0.15±0.088rem/mCi), adrenals (0.14±0.027 rem/mCi), heart wall (0.085±0.014rem/mCi), osteogenic cells (0.084±0.0048 rem/mCi), and red bone marrow(0.083±0.0099 rem/mCi). The mean whole body dose estimate was0.044±0.00031 rem/mCi, and the mean effective dose as defined in ICRP 60was 0.070±0.0059 rem/mCi. See Example 25 below for more information ofeffective dose.

Based on average values, the maximum dose of imaging agent-1 that may beadministered to a human without exceeding 50 mSv (5 rem) to the urinarybladder was estimated to be 12 mCi. Similarly, the maximum administereddose that does not exceed 10 mSv effective dose was estimated to be 14mCi.

Example 24

The follow Example describes the organ bio-distribution and dosimetryfor imaging agent-1.

Whole organ bio-distribution and dosimetry for ¹⁸F-labeled imagingagent-1 were determined based on PET image data from twelve healthysubjects. Image quantification, kinetic modeling to determine residencetimes, and dosimetry analysis were performed.

Head to mid-thigh PET image data for twelve healthy subjects wereobtained using ¹⁸F labeled imaging agent-1 at approximately 17, 31, 45,117, 190, and 225 minutes post injection. Additionally, leg images werealso obtained at approximately 66 and 274 minutes post injection. Imagedata were attenuation corrected at the imaging site, and were quantifiedbased on the Medical Internal Radiation Dosimetry (MIRD) 16 methodologyto determine kinetic data in all organs showing significant uptake ofactivity. Dosimetry estimates were created via kinetic modeling of thequantified image data to determine residence times, and the standardMIRD methodology using a method similar. Kinetic data, residence times,and the dosimetry estimates were reported for each subject and assummary statistics.

Results

No adverse events due to imaging agent-1 were observed. Approximately1.6% of the injected dose (ID) was seen in the myocardium initially,remaining above 1.5% of ID (decay-corrected) through 4 hours afterinjection. The ratio of myocardial to liver radioactivity wasapproximately one initially increasing to more than two at 4 hours.Blood radioactivity cleared quickly, and lung activity was lowthroughout the study. On average, the organ that showed the largest peakuptake was the urinary bladder with approximately 18.3% of the injectedactivity. The next largest peak uptake occurred in the liver withapproximately 15.5% of the injected activity.

Dosimetry Estimates:

On average, the organ receiving the largest absorbed dose was theurinary bladder wall at 0.38 rem/mCi (0.10 mSv/MBq) followed by thekidneys at 0.31 rem/mCi (0.083 mSv/MBq). The mean ED (effective dose)was 0.096 rem/mCi (0.026 mSv/MBq). Table 9 shows the absorbed dosesummary statistics in rem/mCi for all subjects. Table 10 shows theabsorbed dose summary statistics in mGy/MBq for all subjects.

Terms:

The following terms are used in connection with this Example.

Effective Dose (ED): Developed by the ICRP for occupational radiationprotection, the ED enables the comparison of radiation detriment from auniform external dose and a non-uniform internal dose. The risk for a 1rem ED determined for a non-uniform internal dose is equal to the riskfrom a 1 rem uniform external exposure (total body dose). As defined inICRP publication 60 [ICRP-60 1991].

Effective Dose Equivalent (EDE): Developed by the InternationalCommission on Radiological Protection (ICRP) for occupational radiationprotection, the EDE enables the comparison of radiation detriment from auniform external dose and a non-uniform internal dose. The risk for a 1rem EDE determined for a non-uniform internal dose is equal to the riskfrom a 1 rem uniform external exposure (total body dose). As defined inICRP publication 30 [ICRP-30 1981].

TABLE 9 All Subjects - Absorbed Dose Estimates (rem/mCi) n = 12 StandardMean Deviation Min Max Adrenals 0.051 0.003 0.045 0.056 Brain 0.0190.003 0.017 0.026 Breasts 0.024 0.002 0.022 0.029 Gallbladder Wall 0.0590.005 0.050 0.069 LLI Wall 0.047 0.003 0.041 0.051 Small Intestine 0.1700.029 0.121 0.215 Stomach Wall 0.114 0.028 0.088 0.193 ULI Wall 0.0590.005 0.051 0.066 Heart Wall 0.105 0.016 0.083 0.146 Kidneys 0.309 0.0520.225 0.387 Liver 0.141 0.039 0.092 0.229 Lungs 0.108 0.019 0.075 0.146Muscle 0.030 0.002 0.028 0.035 Ovaries 0.053 0.003 0.046 0.057 Pancreas0.050 0.003 0.044 0.057 Red Marrow 0.072 0.009 0.056 0.090 OsteogenicCells 0.060 0.005 0.049 0.069 Salivary Glands 0.127 0.053 0.075 0.280Skin 0.020 0.002 0.019 0.025 Spleen 0.111 0.029 0.072 0.165 Testes 0.0270.002 0.025 0.031 Thymus 0.029 0.003 0.027 0.036 Thyroid 0.243 0.0390.172 0.294 Urinary Bladder Wall 0.376 0.073 0.179 0.463 Uterus 0.0620.003 0.057 0.068 Total Body 0.038 0.002 0.036 0.043 EDE 0.115 0.0060.103 0.121 ED 0.096 0.005 0.090 0.107

TABLE 10 All Subjects - Absorbed Dose Estimates (mSv/MBq) n = 12Standard Mean Deviation Min Max Adrenals 0.0138 0.0009 0.0121 0.0152Brain 0.0052 0.0007 0.0046 0.0069 Breasts 0.0065 0.0006 0.0060 0.0079Gallbladder Wall 0.0159 0.0014 0.0136 0.0187 LLI Wall 0.0128 0.00070.0112 0.0137 Small Intestine 0.0460 0.0079 0.0327 0.0581 Stomach Wall0.0308 0.0077 0.0238 0.0520 ULI Wall 0.0159 0.0013 0.0136 0.0178 HeartWall 0.0285 0.0043 0.0223 0.0395 Kidneys 0.0834 0.0141 0.0608 0.1046Liver 0.0382 0.0104 0.0249 0.0619 Lungs 0.0291 0.0053 0.0201 0.0395Muscle 0.0081 0.0005 0.0077 0.0095 Ovaries 0.0143 0.0009 0.0123 0.0155Pancreas 0.0136 0.0009 0.0120 0.0155 Red Marrow 0.0196 0.0023 0.01500.0242 Osteogenic Cells 0.0163 0.0015 0.0133 0.0187 Salivary Glands0.0343 0.0144 0.0204 0.0758 Skin 0.0055 0.0005 0.0051 0.0068 Spleen0.0300 0.0080 0.0195 0.0446 Testes 0.0074 0.0005 0.0067 0.0085 Thymus0.0080 0.0007 0.0074 0.0098 Thyroid 0.0657 0.0106 0.0465 0.0795 UrinaryBladder Wall 0.1015 0.0197 0.0484 0.1251 Uterus 0.0169 0.0009 0.01550.0183 Total Body 0.0104 0.0004 0.0098 0.0115 EDE 0.0309 0.0015 0.02780.0327 ED 0.0260 0.0012 0.0244 0.0288

These data showed that imaging agent-1 was well tolerated and yielded aradiation dose comparable to that of other commonly-used PETradiopharmaceuticals. Myocardial uptake and adjacent organ activityshowed that it was possible to acquire good images with acceptablepatient radiation dose.

Example 25

Imaging agent-1 was designed as a substrate for the norepinephrinetransporter (NET) to image the cardiac sympathetic nervous system.Competition experiments using cell membranes over-expressing the humanNET indicated a K_(i) value of 5.16±0.93 μM. In a human neuroblastomacell line (SH-SY5Y), uptake of imaging agent-1 was inhibited bydesipramine, a selective NET inhibitor, and the uptake kinetics wasdetermined with K_(m) and V_(max) values of 6.78±1.94 μM and 5.18±1.23μmol/min/million cells, respectively. These values were similar to thatof MIBG (2.12±0.26 μM and 4.76±0.78 μmol/min/million cells). In animals,tissue biodistribution of imaging agent-1 was assessed by tissuesampling at 15- and 60-minute following administration. Heart uptake was2.36±0.16 and 2.17±0.12% injected dose per g tissue (% ID/g) in rats and0.25±0.03 and 0.28±0.03% ID/g in rabbits. In rabbits, desipramine (1mg/kg) inhibited heart uptake of imaging agent-1 by 68% and ¹²³I-MIBGuptake by 55% at 1 hour post dose. Furthermore, sympathetic denervationwith 6-hydroxydopamine (6-OHDA, i.v.) also resulted in a marked decreasein imaging agent-1 uptake in the heart by 79%. Cardiac imaging withimaging agent-1 consistently showed clear myocardium with minimalbackground interference from blood, lung, or liver in rats, rabbits, andnonhuman primates (NHP). Consistent with biodistribution studies,imaging studies in rabbits, pretreatment with desipramine demonstratedreduced levels of radioactivity in the heart in a dose dependent manner.Similarly, 6-OHDA induced sympathetic denervation resulted in lowcardiac image intensity with imaging agent-1 but normal perfusion imageswith the PET perfusion agent,(2-tert-butyl-4-chloro-5-[4-(2-[¹⁸F]fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1(see International PCT Publication No. WO2005/079391, published Sep. 1,2005, incorporated herein by reference). Cardiac imaging with imagingagent-1 in NHPs pretreated with desipramine (0.5 mg/kg) showed adecreased radioactivity in the heart by 68%. Collectively, in vitro andin vivo findings indicate that imaging agent-1 may be used as a cardiacPET imaging agent transported into the heart via NET and may be used forassessment of cardiac neuronal function.

Example 26

The prognostic value of imaging agent-1 was evaluated in Dahl SaltSensitive (DSS) rats, a rat model of heart failure (HF), and comparedwith ¹²³I-meta-iodobenzylguanidine (¹²³I-MIBG). DSS rats were fed eithera low salt (0.1% as control) or high salt diet (8%) for 5 or 9 weeks. Todetermine the progression of HF in these rats, plasma norepinephrinelevels, and heart and lung weights were measured. Compared to low saltdiet groups, DSS rats fed a high salt diet for 5 weeks had markedincreases in norepinephrine levels (258±28 vs. 1242±184 pg/mL) and heartto body weight ratio (3.3±0.1 vs. 4.5±0.3 mg/g). By 9 weeks, thenorepinephrine levels (656±219 vs. 1508±165 pg/mL) and heart to bodyweight ratio (3.2±0.1 vs. 6.1±0.3 mg/g) had increased further and thelung to body weight ratio had become elevated (3.9±0.1 vs. 14.0±1.4mg/g). These rats fed a high salt diet were demonstrated to develop HFfrom early stage HF with myocardial hypertrophy (5-week) to late stageHF with sever lung congestion (9-week). Imaging agent-1 and MIBG heartuptake was examined in early and late stage HF rats by tissue samplingafter intravenous administration. The uptake was measured using a gammacounter and expressed as differential absorption ratio (DAR). Theimaging agent-1 heart uptake decreased following progression of HF fromearly to late stage HF (low salt group vs. high salt group: 6.9±0.6 vs.5.1±0.6 and 8.1±0.2 vs. 3.1±0.2 DAR at 5 and 9 weeks respectively).These findings were comparable with the heart uptake of ¹²³I-MIBG inthese rats (7.3±0.1 vs. 3.8±0.5 and 7.9±0.5 vs. 2.3±0.3 DARrespectively). Cardiac PET imaging with imaging agent-1 in DSS rats feda low salt diet showed clear myocardium with minimal backgroundinterference from blood, lung, and liver. Consistent with the findingsin the tissue sampling, imaging in DSS rats fed a high salt diet showedprogressively reduced radioactivity in the heart of these rats from 5 to9 weeks. These results suggest that the profile of imaging agent-1 issimilar to ¹²³I-MIBG, and cardiac imaging with imaging agent-1 can beused to detect progression of HF in DSS rats.

Example 27

Imaging with ¹²³I-meta-iodobenzylguanidine (MIBG) has been shown topredict heart failure progression, but the image quality is poor. LikeMIBG, imaging agent-1 was designed as a substrate for norepinephrinetransporter (NET), but labeled with ¹⁸F to take advantages of PETtechnology. This study evaluated cardiac image quality of imagingagent-1 and its affinity and selectivity to NET and uptake kinetics, incomparison with norepinephrine (NE).

Methods:

The affinity (K_(i)) was determined in a competition binding assay byincubating ¹⁹F-imaging agent-1, a cold analog of imaging agent-1, or NEwith ³H-desmethylimipramine in cell membrane overexpressing human NET.The uptake selectivity was assessed by measuring imaging agent-1 or³H-NE cell uptake with and without pretreatment of desipramine, aselective NET inhibitor, in SK—N—SH (human neuroblastoma) and PC-12 (ratpheochromocytoma) cells. In SK—NSH cells, the uptake kinetics (K_(m) andV_(max)) were evaluated by measuring NET mediated uptake of imagingagent-1 or NE at various concentrations. Imaging agent-1 cardiac imagequality was evaluated by PET imaging (˜1.5 mCi, i.v.) in rabbits in thepresence and absence of desipramine (1 mg/kg).

Results: In competition binding assay, K_(i) values for imaging agent-1and NE were similar (5.2±1.1 and 3.41±0.3 μM). In cell studies, blockadeof NET inhibited imaging agent-1 and NE uptake by 66±7 and 931% in PC-12cells, and 911 and 97±1% in SK—N—SH cells. In SK—N—SH cells, K_(m) andV_(max) values for imaging agent-1 were 1.4±0.3 μM and 6.0±1.3pMol/million cells/min similar to that of NE (2.0±0.4 μM and 6.2±0.7pMol/million cells/min). Moreover, imaging agent-1 cell uptake wasinhibited by imaging agent-1 or NE concentration-dependently. Imaging inrabbits with imaging agent-1 showed clear myocardium uptake with lowliver activity. Cardiac uptake could be inhibited by desipramine.

The cell uptake profile of imaging agent-1 was similar to NE with highselectivity. Cardiac images of imaging agent-1 were clear, and the heartuptake was mediated by NET.

Example 28

Cardiac sympathetic denervation (CSD) assessed by¹²³I-metaiodobenzylguanidin (MIBG) imaging has been suggested to predictcardiac events including arrhythmia and death in heart failure patients(ADMIRE-HF trial). This study evaluated imaging with imaging agent-1 toidentify CSD.

Methods:

Rabbit models of regional and systemic CSD were used. To developregional CSD, a median sternotomy was performed and phenol (89% inliquid) was pained on the anterior and posterior walls of the leftventricle. To develop systemic denervation the neurotoxin,6-hydroxydopamine (25 mg/kg on day 1, 2, 7 and 8), was administeredintravenously. Two weeks following these procedures, rabbits were imagedwith imaging agent-1 (˜1.5 mCi, i.v.) using a microPET camera for 30minutes. To ensure the denervation procedures did not result inperfusion changes rabbits were also imaged with the ¹⁸F perfusionimaging agent(2-tert-butyl-4-chloro-5-[4-(2-[¹⁸F]fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1.

Results:

In sham-denervated rabbits, cardiac images of imaging agent-1 showedclear myocardium with uniform radioactivity distribution. Theradioactivity was low in the lung and liver and cleared rapidly inblood. In rabbits with systemic denervation, image based quantificationindicated an ˜80% global reduction in heart uptake of imaging agent-1compared to control animals. Similarly, regional denervation resulted ina marked reduction in imaging agent-1 in the treated regions. Incontrast, cardiac imaging with(2-tert-butyl-4-chloro-5-[4-(2-[¹⁸F]fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1demonstrated well-perfused myocardium, and no differences were observedbetween control and denervated rabbits.

Reduced imaging agent-1 heart uptake in CSD rabbits was found to be dueto impaired innervation, not to alterations in perfusion. Cardiac PETimaging with imaging agent-1 was used for detection of CSD, like¹²³I-MIBG, but with improved image quality and quantification.

Example 29

The following Example describes roles of cardiac norepinephrine uptake 1and 2 in evaluation of imaging agent-1 in rats, rabbits, and non-humanprimates

Objectives:

Norepinephrine (NE) released from cardiac sympathetic nerves issubstantially cleared by neuronal uptake 1 (NE transporter) in rabbits,non-human primates, and humans, and by uptake 1 and 2 in rats. Imagingagent-1 is designed, in part, as a substrate for uptake 1 like NE and¹²³I-meta-iodobenzylguanidine (MIBG). This study examined speciesdifferences associated with cardiac uptake 1 and 2 for cardiac uptake ofimaging agent-1.

Methods:

Desipramine, a selective uptake 1 inhibitor, was used to block cardiacuptake 1 in rats (10 mg/kg, ip), rabbits (1 mg/kg iv), and NHPs (0.5mg/kg, iv). 6-hydroxydopamine, a neurotoxin, was injected to inducesympathetic denervation in rats (100 mg/kg ip for 7 days) and rabbits(25 mg/kg iv on day 1, 2, 7, and 8). Imaging agent-1 heart uptake incomparison with MIBG was assessed by tissue sampling at 60 minutes postimaging agent injection. Imaging was also performed with(2-tert-butyl-4-chloro-5-[4-(2-[¹⁸F]fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1.

Results:

In rats, blockade of uptake 1 did not alter imaging agent-1 heart uptakecompared to the control (1.41±0.07 vs. 1.47±0.22% injected dose per gramof tissue (% ID/g)). In contrast, imaging agent-1 heart uptake wasreduced by 68% in uptake 1 blocked rabbits. In sympathetic denervatedrats, imaging agent-1 heart uptake was comparable to the control group(2.18±0.39 vs. 2.58±0.76% ID/g). However, the uptake decreased markedly(79%) in sympathetic denervated rabbits. Similar results were found inMIBG heart uptake in rats and rabbits with uptake 1 blockade andsympathetic denervation. Consistently,(2-tert-butyl-4-chloro-5-[4-(2-[¹⁸F]fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1cardiac imaging showed comparable myocardial activity in sympatheticdenervated rats to the control, but marked activity reduction indenervated rabbits and uptake 1 blocked rabbits and NHPs.

Conclusions:

In rabbits and non-human primates with uptake 1 as the main cardiac NEtransporter, similar to humans, imaging agent-1 demonstrated highselectivity to neuronal uptake 1 and can be used in evaluation ofcardiac sympathetic denervation. Due to high cardiac expression ofuptake 2, in some embodiments, evaluation of uptake 1 substrate basedneuronal imaging agents in rats should be done with caution.

Example 30

The following Example describes the assessment of heart failuremedications on cardiac uptake of imaging agent-1.

Objectives:

This study investigated if commonly used HF medications affect NETmediated imaging agent-1 uptake.

Methods:

NET mediated uptake of imaging agent-1 was detected in SK—N—SH cells(human neuroblastoma known to express NET) by incubating 1 million cellswith the ligand for 60 minutes in the presence or absence of desipramine(1 μM), a selective NET inhibitor. To assess drug impact on imagingagent-1 uptake, cells were pre-incubated (15 minutes) with vehicle orvarious concentrations (0.001 to 1000 μM) of propranolol (receptorblocker), captopril (ACE inhibitor), losartan (angiotensin II receptorinhibitor), or verapamil (calcium channel blocker) before addition ofimaging agent-1 (1 μCi).

Results:

Imaging agent-1 cell uptake was 26±2% in SK—N—SH cells, and the majority(88%) was inhibited by desipramine. Substantial reduction of imagingagent-1 uptake was only observed by pre-incubation with propranolol atconcentrations above 1 μM and with verapamil at concentrations above 10μM. Losartan and captopril had no effect on imaging agent-1 uptake evenat the highest concentrations tested (1000 μM) The concentrations ofthese HF medications producing inhibition of imaging agent-1 uptake weresubstantially above the steady state levels achieved for these drugswhen used clinically.

Conclusions:

Based on these in vitro studies, several commonly used HF medications donot inhibit NET mediated imaging agent-1 uptake at clinically relevantconcentrations.

Example 31

The following Example describes the evaluation of cardiac denervation,re-innervation and associated susceptibility to arrhythmia using imagingagent-1.

Objectives:

Regional cardiac sympathetic denervation (RCSD) may be associated withcardiac arrhythmia in heart failure patients. This study evaluatedwhether imaging agent-1 imaging could be used to measure RCSD subsequentre-innervation and potential association with arrhythmia susceptibility.

Methods:

Rabbit models of RCSD were developed by applying phenol directly on thesurface of the left ventricular wall during a sternotomy. Two and twelveweeks following the procedure, imaging agent-1 cardiac PET imaging (˜1.5mCi, iv) were performed in these rabbits. The myocardial area withradioactivity ≧50% maximum was quantified as innervated region forcomparison. To evaluate susceptibility of arrhythmia in rabbits,dofetilide (10 and 40 μg/kg iv, a delayed I_(Kr) inhibitor) inducedchanges were assessed by measuring ECG including heart rate (HR), QTcinterval (corrected by Fridericia method), and frequency of arrhythmia.

Results:

Cardiac images showed clear homogeneous myocardial uptake of imagingagent-1 in sham-denervated rabbits and reduced levels in phenol RCSDinduced rabbits at 2 weeks post surgery (20702±2190 vs. 12245±905 voxelsrespectively). The denervated region was reduced by 12 weeks (16812±503voxels) indicating re-innervation. Imaging with(2-tert-butyl-4-chloro-5-[4-(2-[¹⁸F]fluoroethoxymethyl)-benzyloxy]-2H-pyridazin-3-1,a PET perfusion imaging agent, showed homogeneous myocardialdistribution in all rabbits including those with RCSD regions indicatingdenervation did not alter blood flow. Dofetilide induced QTcprolongation, frequency of premature ventricular contraction, andtorsades de pointes were more prominent in the RCSD group than incontrol. However, the changes in HR were comparable in the two groups.

Conclusions:

Imaging agent-1 cardiac imaging detected RCSD and re-innervation. TheRCSD increased susceptibility of drug induced QTc prolongation andarrhythmia.

Example 32

The following Example describes evaluation of imaging agent-1 intumor-bearing mice.

Example 32A Preparation of Tumor Bearing Mouse Models

Xenograft Model:

Four to six week-old female Nude mice were anesthetized to immobilizethem for subcutaneous inoculation with a range of 1.0×10⁶/0.1mL-1.0×10⁸/0.1 mL cells in sterile cell culture media then returned totheir cages to recover. Cell lines were co-injected with a commerciallyavailable growth matrix (50/50 v/v) to facilitate tumor development(Matrigel®-BD Bioscience). Human cell lines included PC12(pheochromocytoma), SH—SY-5Y and SK—N—SH (neuroblastoma).

Oncomouse Model:

Obtained through in-house breeding program.

Example 32B Tissue Biodistribution

Tumor bearing mice (100-1500 mm³ tumor size) were anesthetizedintramuscularly with 0.1 mL of ketamine/acepromazine (1.8 mL saline, 1.0mL ketamine, and 0.2 mL acepromazine) prior to dosing and tissuesampling. Individual mice were then injected via the tail vein withimaging agent-1 (0.5-2.0 mCi/kg in 0.1 mL). Mice were euthanized andbiodistribution performed at 1 h post-injection. Selected tissues wereremoved, weighed, and counted on a gamma counter. Results are expressedas the percentage injected dose per gram of tissue (% ID/g; FIG. 10).Since the c-neu Oncomouse® spontaneously develops tumors in the mammaryglands, most mice had more than one tumor. Each tumor was sampled andcounted separately, and radioactive uptake for the tumors was averagedto obtain an overall representation of tumor uptake. Xenograft mice hadonly one tumor implanted and harvested at the time of tissuedistribution analysis.

Terms and Equivalents

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1-102. (canceled)
 103. A salt of formula (VII):

wherein X^(⊖) is ascorbate, optionally, wherein the fluorine isisotopically enriched with ¹⁸F, or a salt of formula (VI):

wherein X^(⊖) is formate, optionally, wherein the fluorine isisotopically enriched with ¹⁸F.
 104. A pharmaceutically acceptablecomposition comprising a salt of claim 103, and optionally apharmaceutically acceptable excipient.
 105. A composition comprising thesalt of claim 103 for imaging a portion of a subject.
 106. Use of thesalt of claim 103, in the preparation of a medicament for imaging atleast a portion of the subject, wherein the salt is provided in a doseof 15 mCi or less, 14 mCi or less, or 13 mCi or less.
 107. Use of thesalt of claim 103, in the preparation of a medicament for detectingnorepinephrine transporter (NET) in a portion of a subject, wherein thesalt is provided in a dose of less than 14 mCi.
 108. The use of claim107, wherein dose is 13 mCi or less, is between 10 mCi and 13 mCi, or isbetween 8 mCi and 10 mCi.
 109. The use of claim 107, wherein the portionof the subject is at least a portion of the cardiovascular system, atleast a portion of the heart, or at least a portion of a tumor.